Synthesis of dimeric, trimeric, tetrameric pentameric, and higher oligomeric epicatechin-derived procyanidins having 4beta,8-interflavan linkages and their use to inhibit cancer cell growth through cell cycle arrest

ABSTRACT

Various processes are disclosed for preparing protected epicatechin oligomers having (4β,8)-interflavan linkages. In one process, a tetra-O-protected epicatechin monomer or oligomer is coupled with a protected, C-4 activated epicatechin monomer in the presence of an acidic clay such as a mortmorillonite clay. In another process, a 5,7,3′,4′-benzyl protected or a 3-acetyl-, 5,7,3′,4′-benzyl protected epicatechin or catechin monomer or oligomer is reacted with 3-O-acetyl-4-[(2-benzothiazolyl)thio]-5,7,3′,4′-tetra-O-benzylepicatechin in the presence of silver tetrafluoroborate. In another process, two 5,7,3′,4′-benzyl protected epicatechin monomers activated with 2-(benzothiazolyl)thio groups at the C-4 positions are cross-coupled in the presence of silver tetrofluoroborate. A process is also disclosed for reacting an unprotected epicatechin or catechin monomer with 4-(benzylthio) epicatechin or catechin. The use of naturally-derived and synthetically-prepared procyanidin (4β,8) 4 -pentamers to treat cancer is also disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional ApplicationNo. 60/415,616 filed Oct. 2, 2002 and entitled “Synthesis of Dimeric,Trimeric, Tetrameric, Pentameric, and Higher OligomericEpicatechin-Derived Procyanidins Having 4β,8-Interflavan Linkages andTheir Use To Inhibit Cancer Cell Growth through Cell Cycle Arrest”.

BACKGROUND OF THE INVENTION

[0002] Condensed tannins (proanthocyanidins) are widespread in the plantkingdom, form part of the human diet, and display multiple biologicalactivities that render them significant to health. Procyanidins haveattracted a great deal of recent attention in the fields of nutrition,medicine and health due to their wide range of potentially significantbiological activities. There is a growing body of evidence suggestingthat these compounds act as potent antioxidants in vitro, ex vivo and invivo and may thus alter the pathophysiology of imbalances orperturbations of free radical and/or oxidatively driven processes inmany diseases or directly interfere with many cellular processes. SeeNijveldt, R. J. et al., Am. J. Clin. Nutr. 2001, 74, 418. Initialobservations also have shown that procyanidin-rich fractions extractedfrom defatted cocoa beans elicited in vitro growth inhibition in severalhuman cancer cell lines. See U.S. Pat. No. 5,554,645 issued Sep. 10,1996 to L. J. Romanczyk, Jr. et al.

[0003] Isolation, separation, purification, and identification methodshave been established for the recovery of a range of procyanidinoligomers for comparative in vitro and in vivo assessment of biologicalactivates and currently some oligomers can be synthesized usingtime-consuming method. For instance, previous attempts to couplemonomeric units in free phenolic form using mineral acid as the catalystin aqueous media have met with limited success. The yields were low, thereactions proceeded with poor selectivity, and the oligomers weredifficult to isolate. See Steynberg, P. J., et al., Tetrahedron, 1998,54, 8153-8158. An overview of the shortcomings is set out below.

[0004] Benzylated monomers were prepared using benzyl bromide incombination with potassium carbonate (K₂CO₃) and dimethyl formamide(DMF). See Kawamoto, H. et al., Mokuzai Gakkashi, 1991, 37, 741-747. Theyield, however, was only about 40%. In addition, competing C-benzylationleads to a mixture of products, which make isolation of thebenzyl-protected target monomer more difficult. Also, partialracemization of (+)-catechin at both the C-2 and C-3 positions wasobserved (see Pierre, M.-C. et al., Tetrahedron Letters, 1997, 38, 32,5639-5642).

[0005] Two primary methods for oxidative functionalization are taught inthe literature. See Betts, M. J. et al., J. Chem. Soc., C, 1969, 1178and Steenkamp, J. A., et al., Tetrahedron Lett., 1985, 3045-3048. In theolder method, protected (+)-catechin was treated with lead tetraacetate(LTA) in benzene to produce the 4β-acetoxy derivative which was thensuccessfully hydrolyzed to the 3,4-diol. Flavan-3,4-diols are incipientelectrophiles in the biomimetric synthesis of procyanidins. However,flavan 3,4-diols which have an oxygen functionality at the C-4 positionare not available from natural sources and have to be synthesized.Oxidative functionalization of the prochiral benzylic position in thesynthesis of procyanidins. The major drawback of this reaction was a lowyield (30-36%) of the acetate during the LTA oxidation. The more recentmethod of oxidatively functionalizing the C-4 position relies on the useof 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). In this method, theprotected monomer was treated with DDQ in methanol. This allowsintroduction of a methoxy group at the C-4 position in a stereospecificmanner. The yield is about 40-50%.

[0006] There are a number of reports on the coupling reaction betweenmonomers and their 3,4-diols in aqueous acid. These methods areunsatisfactory because the low yields, lack of specificity, anddifficulty in the purification from aqueous media. See Kawamoto, H. etal., J. of Wood Chem. Tech., 1989, 9, 35-52 who report the titaniumtetrachloride (TiCl₄) mediated coupling between 4-hydroxyltetra-O-benzyl (+)-catechin and 5 equivalents (eq.) oftetra-O-benzyl(+)-catechin to produce a 3:2 mixture of 4α,8 and 4β,8catechin dimers. This coupling leads to the 4β,8-dimer together withhigher oligomers in yields that decrease with the increasing molecularmass of the oligomer.

[0007] Using a 2,3-cis-3,4-trans-flavan-3,4-diol, B₂ and B₅ derivativeswere synthesized. The diol was prepared by the acyloxylation of the C-4benzylic function of an (−)-epicatechin tetramethyl ether with leadtetraacetate in a benzene solution. This oxidative functionalization ofthe C-4 position of the methyl protected epicatechin monomer wasimproved by using 2,3-dichloro-5,6-dicyano-1,4-benoquinone (DDQ) inmethanol to introduce a methoxy group at the C-4 position. The protectedmonomer with C-4 methoxy group was used in the synthesis of (4,8) linearprocyanidin oligomers up to the trimers. See Steenkamp et al., Tetr.Lett. 1985 26, 25, 3045-3048.

[0008] Procyanidin oligomers were prepared using a protected epicatechinor catechin monomer having, as a C-4 acyloxy group, a C₂-C₆ alkoxy grouphaving a terminal hydroxy group such as a 2-hydroxyethoxy group. Theprotecting groups used are those that do not deactivate the A ring ofthe monomer, e.g., benzyl protecting groups. See Kozikowski, A. P. etal. J. Org. Chem. 2000, 65, 5371-5381 and U.S. Pat. No. 6,207,842(issued Mar. 27, 2001 to Romanczyk, L. J. et al.). The C-4 derivatized,protected monomer was coupled with a protected catechin monomer orprotected epicatechin monomer to form a protected 4,8 dimer which wasthen deprotected or used for further coupling with another protected,C-4 derivatized epicatechin monomer to form protected higher 4,8oligomers. If a 4,6 linkage was desired, the C-8 position of theprotected catechin or epicatechin monomer was blocked with a halogengroup prior to coupling with the C-4 derivatized, protected epicatechinmonomer or oligomer. Higher oligomers having both 4,8 and 4,6 linkageswere also be prepared. The protected dimers or oligomers were deblocked,and if necessary, deprotected, e.g., by hydrogenolysis. The coupling wascarried out in the presence of a protic acid or a Lewis acid such astitanium tetrachloride (TiCl₄). The stereochemical nature of theinterflavan bond was confirmed by the synthesis of a specificallyprotected derivative and its subsequent degradation. Furthermore,titanium tetrachloride-mediated chain extension of epicatechin leads tothe formation of regioisomers. This is a serious drawback, not only interms of yield, but also purity. Even though the 4β,8-trimers and4β,8-tetramers were isolated in pure form, the same can notautomatically be expected for the larger oligomers, for which the numberof possible isomers, and thus contaminants, grows rapidly.

[0009] One potential way of dealing with this problem is to carefullypurify the chain-extended oligomer after each step in order to ensurethat all chain-extended oligomers are at least derived from a singleisomer of the starting oligomer. However, upon the titaniumtetrachloride-mediated chain extension of the C-4 derivatized, protectedmonomer with two equivalents of the protected trimer, not only were theprotected tetramer, pentamer, and small amounts of higher oligomersformed, but the protected trimer was degraded to the monomer and dimer,which then participated in the chain-extension reaction, giving rise toregioisomeric oligomers such as small amounts of the protected4β,6:4β,8-trimer. While the reaction conditions (methylenechloride/tetrahydrofuran (9:11), 0° C., 15 min., then room temperature,140 min.) were not optimized, chain degradation warranted a search for abetter synthetic approach.

[0010] Thus, there is a need for improved methods for synthesizingepicatechin oligomers, particularly the higher oligomers, and a processfor using protected larger epicatechin oligomers as building blocks forchain extension to even larger oligomers.

SUMMARY OF THE INVENTION

[0011] In one embodiment, bis(5,7,3′,4′-tetra-O-protected) epicatechin(4β,8)-dimer and higher (4β,8)-oligomers are prepared by coupling a(5,7,3′,4′-tetra-O-protected) epicatechin monomer with a5,7,3′,4′-tetra-O-protected-4-(acyloxy) epicatechin monomer in thepresence of an acidic clay. The benzyl-protected (4β,8)-dimer isproduced in significantly increased yields. Under the same conditions,the benzyl-protected (4β,8)-trimer, -tetramer, and -pentamer areobtained regioselectively from the 5,7,3′,4′-tetra-O-protected(4β,8)-oligomer. The preferred acidic clay is a mortmorillonite clay.The protecting groups used should not deactivate the A ring of theprotected monomers or the A ring of the upper mer of the protectedoligomers. The preferred protecting groups are benzyl groups. A suitable4-acyloxy group is a C₂-C₆ alkoxy group having a terminal hydroxylgroup, preferably 2-hydroxyethoxy. The protected monomer and protecteddimer, as well as the protected oligomers, are separated by columnchromatography and then the protecting groups are replaced withhydrogen.

[0012] In another embodiment, a mixture of benzyl-protected(4β,8)-oligomers of epicatechin or catechin, such as the trimer throughpentamer, are prepared in improved yields by reacting a benzyl-protected(4β,8)-dimer (e.g., bis(5,7,3′,4′-tetra-O-benzyl)epicatechin(4β,8)-dimer or bis(3-O-acetyl-5,7,3′, 4′-tetra-O-benzyl)epicatechin(4β,8)-dimer with a 3,5,7,3′,4′-protected epicatechin monomer having, asa C-4 activating group, a (2-benzothiazolyl)thio group. To avoid theundesired intervention of the 3-hydroxyl group, this group is protectedin both the electrophilic and nucleophilic reaction partners byacetylation. The reaction is carried out in the presence of silvertetrafluoroborate (AgBF₄). Preferably, the silver tetrafluoroborate isdried before the reaction. More preferably the drying is vacuum dryingcarried out immediately before the reaction. The resulting mixturecomprises protected trimers through protected octamers. The protectedoligomers are isolated by reverse phase high pressure liquidchromatography. The acetyl protecting group(s) are removed, preferablywith aqueous tetra-n-butyl ammonium hydroxide. The benzyl protectinggroups are removed by hydrogenolysis, preferably after removal of theacetyl protecting group(s). The yields are near-quantitative. Theoligomers are characterized as their peracetates. The syntheticprocyanidin oligomers are identical to the procyanidin oligomersisolated from cocoa bean extracts by normal-phase HPLC.

[0013] In another embodiment, chain extension by cross-coupling of twobenzyl-protected epicatechin (4β,8)-oligomers each having aC-4-(2-benzylthiazolyl)thio group is carried out in the presence ofsilver tetrafluoroborate.

[0014] A process is provided for preparing the5,7,3′,4′-tetra-O-benzylepicatechin or -catechin monomer having a C-4(2-benzothiazoly)-thio group. The process involves reacting5,7,3′,4′-tetra-O-benzyl-4-(2-hydroxyethoxy)epicatechin or5,7,3′,4′-tetra-O-benzyl-4-(2-hydroxyethoxy)catechin with anorganoaluminum thiolate generated from 2-mercaptobenzothiazole. Aprocess is also provided for preparing3-O-acetyl-4[(2-benzyothiazolyl)thio]5,7,3′,4′-tetra-O-benzylepicatechin or3-O-acetyl-4-[(2-benzothiazolyl)thio] 5,7,3′,4′-tetra-O-benzylcatechinby reacting 5,7,3′,4′-tetra-O-benzyl-4-(2-hydroxyethoxy)epicatechin or5,7,3′,4′-tetra-O-benzyl-4-(2-hydroxyethoxy)catechin with anorganoaluminum thiolate generated from 2-mercaptobenzothiazole followedby acetylation of the C-3 hydroxyl group.

[0015] A process is also provided for exclusively preparing aprocyanidin (4β,8)-dimer by reacting an unprotected epicatechin orcatechin monomer with 4-(benzythio)catechin or 4-(benzylthio)epicatechinin the presence of dimethyl(methylthio)sulfonium tetrafluoroborate orpreferably silver tetrafluoroborate.

[0016] When tested in several breast cancer cell lines, both thesynthetic and natural procyanidin pentamer, and to a lesser extent thetetramer, inhibited cell growth. Using the MDA MB-231 cell line, it wasestablished that this outcome is based on the induction of cell cyclearrest in the G₀/G₁ phase. Subsequent cell death is more likely necroticrather than apoptotic. Control experiments demonstrate that theprocyanidin itself, rather than hydrogen peroxide, is the causativeagent.

[0017] The regio-and stereochemistry of the interflavan linkages hasbeen established by partial thiolysis (see Hor, M. et al.,Phytochemistry 1996, 42, 109). For the tetramer the upper interflavanlinkage is 4β,8 and the lower portion of the molecule is identical tothe trimer which has also been subjected to partial thiolysis with bothlinkages being identified as 4β,8 (see Hor et al.). Since in the courseof the present chain extension process the first three interflavanlinkages formed are exclusively 4β,8 linkages, the same must be true forthe additional interflavan linkages present in the higher oligomers.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] A. Chain Extension of Protected Epicatechin Dimers and TrimersMediated by Acidic Clay

[0019] Chain extension of 5,7,3′,4′-tetra-O-benzylepicatechin mediatedby an acidic clay such as Montmorillomite clay (e.g., Bentonite K-10)results in the almost exclusive formation of the protected 4β,8)-dimerin a 90% isolated yield together with small amounts of the protected(4β,8)₂-trimer. No 4,6-linked oligomers are observed. The surprisinglyhigh reactivity differential under these conditions between the monomerand the dimer allows most of the dimer to survive without entering intofurther chain extension. Reaction of bis(5,7,3′,4′-tetra-O-benzyl)epicatechin (4β,8)-dimer with 5,7,3′,4′-tetra-O-benzylepicatechinactivated at the C-4 position with a 2-hydroxyethoxy group yields 40% ofthe (4β,8)₂-trimer together with 13% of the (4β,8)₃-tetramer by thischain extension protocol.

[0020] The cleanness of this reaction permits, for the first time, atleast a partial separation of the monomer from the dimer, and even ofthe dimer from the trimer, by column chromatography. This significantlyreduces the amount of material that needs to be put through HPLCpurification.

[0021] A simplified procedure for isolating the electrophilic monomericbuilding block having the C-42-hydroxyethoxy group is provided.

[0022] B. Chain Extension of 5,7,3′,4′ Protected or3,5,7,3′,4′-Protected Epicatechin or Catechin Monomers HavingC-4[2-(Benzothiazolyl)thio] Group

[0023] In an alternative chain extension, a 5,7,3′,4′-protectedepicatechin or catechin activated at the C-4 position withmercaptobenzothoiazolyl is used for the chain extension. The chainextension is mediated by silver tetrafluoroborate. The reagent used tointroduce a 2-(benzothiazolyl)thio group at the C-4 position of anepicatechin or a catechin monomer is 2-mercaptobenzothiazole which is anon-volatile odorless heterocyclic thiol. For this coupling, theC-5,7,3′,4′-benzyl-protected monomers, rather than the unprotectedmonomers, are preferred because they are easier to handle, more stable,and more reactive due to the poor accessibility of the unprotected, C-4derivatized monomers.

[0024] The C-4 activated monomer is prepared by reacting a protectedepicatechin or catechin having a 2-hydroxyethoxy group at the C-4position with an organoaluminum thiolate prepared in situ from2-mercaptobenzothiazole and trimethylaluminum. See Dzhemilev U. M. etal., Izv. Akad. Nauk SSSR, Ser. Khim., 1988, 2645. The resulting4-thioether is as a mixture of two stereoisomers which are isolated byfractional crystallization and column chromatography. Only the majorstereoisomer is used for the subsequent coupling.

[0025] The chain extension is effected by adding silver tetrafluorideborate (AgBF₄) to a solution of 5,7,3′,4′-tetra-O-benzylepicatechin or5,7,3′,4′-tetra-O-benzylcatechin and the major stereoisomer. Thisresults in the formation of a protected epicatechin (4β,8)-dimer and anepicatechin (4β,8)₂-trimer. After reverse-phase HPLC separation, theprotected dimer, trimer, and monomer are recovered. Further, separationby reverse-phase HPLC yields, as a by-product, a protected 3-O-4 dimer.To avoid the undesired reaction of the 3-hydroxyl group in this chainextension process, the 3-hydroxyl group is protected by acetylation ofboth the benzyl-protected monomer and the benzyl-protected dimer. Theyields are near-quantitative. When a solution of silvertetrafluoroborate is added to solution of the acetyl-andbenzyl-protected dimer and acetyl- and benzyl protected monomer, theexpected benzyl-protected, acetyl-protected trimer and tetramer areformed, but only in low yields. However, the chain extension proceeds soslowly that adventitious water successfully competes with the flavanoidnucleophile with the major product of the coupling being the 4-hydroxymonomer 3-O-acetyl-5,7,3′,4′-tetra-O-benzyl-4-hydroxyepicatechin. Inaddition, small amounts of the 4-hydroxy dimer(3-O-acetyl-5,7,3′,4′-tetra-O-benzyl-4β,8-(3-O-acetyl-5,7,3′,4′-tetra-O-benzyl-4-hydroxyepicatechin)are also isolated, indicating to self-condensation of the thioetherfollowed by either chain extension or hydrolysis.

[0026] In an attempt to improve the yield, the protected dimer andprotected monomer are dried by stirring with powdered molecular sievesprior to the addition of the silver tetrafluoroborate. The reactants,however, are recovered unchanged. Vacuum drying the silvertetrafluoroborate immediately before the coupling reduces the hydrolysisof3-O-acetyl-4-[(2-benzothiazolyl)thio]-5,7,3′,4′-tetra-O-benzylepicatechinto an acceptable level.

[0027] Using dry silver tetrafluoroborate with a protected monomer toprotected dimer molar ratio of 1:2.5, a series of protected oligomersspanning from the trimer tris(3-O-acetyl-5,7,3′,4′-tetra-O-benzyl)epicatechin (4β,8)₂-trimer to theoctamer octakis (3-O-acetyl-5,7,3′,4′-tetra-O-benzyl)epicatechin(4β,8)₇-octamer can be isolated in a combined yield of 91%. The reactionis exceptionally clean and no 4,6-oligomers are observed.

[0028] Similar results are obtained in the coupling of the C-4derivatized, benzyl- and acetyl-protected monomer with the benzyl-andacetyl-protected trimer and tetramer. From this reaction, oligomers upto the protected undecamer can be isolated by reverse-phase HPLC ifethyl acetate (a nonpolar solvent) is admixed with the acetonitrile inthe final step of the gradient. The use of the ethyl acetate permitsrecovery of the highly retained higher oligomers; however, it alsoelutes significant amounts of aliphatic impurities which subsequentlyhave to be removed by additional HPLC steps, thus reducing the totalproduct recovery.

[0029] All of the protected oligomers (i.e., the benzyl ether-acetates)up to the nonamer were deacetylated in near-quantitative yield with 40%aqueous tetra-n-butylammonium hydroxide in tetrahydrofuran. This base isused because of its good solubility in the relatively nonpolar reactionmedium that is required by the lipophilicity of the starting materials.The ¹H NMR spectra of the resulting benzyl-ethers displayed signals oftwo major rotamers together with trace amounts of additional rotamersthat increase as the oligomeric chain grows. It is believed that theseminor components are rotamers rather than regioisomers because similarsignals are absent from the spectra of the precursor acetates. Samplesof the benzyl-ethers prepared in CDCl₃ exhibit well-resolved,characteristic signals for the hydroxyl (OH) protons in the δ 1.8-1.1region.

[0030] The benzyl-ethers (trimer through the octamers) are deprotectedby hydrogenolysis over Pearlman's catalyst to form the unprotectedoligomers. Preferably, this deprotection is carried out inbicarbonate-washed glassware, as partial fragmentation to loweroligomers is occasionally observed without this precaution, quiteprobably as a consequence of an acidic reaction with the glass surfaceof the reaction flask. To obtain a readily soluble procyanidin, it isnecessary, as similarly reported by others, to dilute the filteredsolution of the crude product with water, evaporate only partially so asto remove most of the organic solvents, and lyophilize the residualsolution. If the crude solutions are directly evaporated to dryness,partially insoluble materials result, indicating that some decompositionhas occurred. Combustion analyses shows that the lyophilized productscontain 1.3-2 equivalents of water per epicatechin moiety.

[0031] Comparison normal phase HPLC analysis of epicatechin(4β,8)₂-trimer, epicatechin (4β,8)₃-tetramer, and epicatechin(4β,8)₄-pentamer was made against the natural trimer, tetramer, andpentamer purified from Theobroma Cocao. Purities ranging from 94% to 96%were observed for the synthetic procyanidins which were 2-4% higher thanthose for the naturally-derived oligomeric procyanidins. The t_(R)'S ofthe synthetic procyanidins match those observed for the naturaloligomers, thus confirming the epicatechin 4β,8 regio- andstereo-chemistry in the natural cocoa procyanidins. All of the naturalprocyanidins purified from cocoa show impurity peaks preceding andfollowing the main peak, with the tetramer and pentamer showing moreimpurity peaks. Scanning these regions by HPLC/MS reveals no change inthe [M]⁺ or [M+Na]⁺ ions indicating that these minor impurities areisomers of the major oligomers. These minor impurities may contribute toin vitro and in vivo activities reported in the literature andpotentially confound structure-activity relationships based only onnatural oligomers. Hence, as a precaution, both natural and syntheticprocyanidins are therefore used in the biological assays reported in thefollowing examples.

[0032] The nature of the impurities and of the side reaction(s) leadingto them has not been established but several trace impurities arepresent rather than a single major one. This is less than ideal;however, comparison of reported optical rotations, for example, of thefree (4β,8)-dimer or the-tetramer reveals large variations that cannotmerely be the consequence of differential degrees of hydration, butappear to indicate the presence of unknown impurities in some of thesesamples as well.

[0033] Since free polyphenols are inherently poorly amenable topurification because of their oxygen and acid sensitivity (acid beingrequired as a solvent additive to reduce peak tailing during HPLC), andtheir NMR spectra are anyway uncharacteristic because of severe linebroadening, these compounds are characterized as their peracetates. Inan attempt to avoid acid-induced interflavan bond cleavage during theacetylation, the amount of pyridine in the acetic anhydride/pyridinereagent can be increased from 1 (the usual amount) to 2 volumes relativeto acetic anhydride, but as a result impurities emerge that eluteimmediately before the peracetates and cannot be removed on apreparative scale. The ¹H NMR spectra of the peracetates exhibit sharpsignals for two rotamers (in a 2;1 ratio for the trimer and in a 3:2ratio for all higher homologs) and are, up to the heptamer or octamer,quite suitable for compound identification, since the acetate regionserves as a useful “fingerprint”. As the oligomeric chain grows, thechemical shift differences between analogous protons of epicatechinunits in the inner position of the chain become eventually insufficientat 300 MHz, resulting in the growth of uncharacteristic signal clusterswithout the appearance of well-separated new signals. These spectra canbe useful for future reference. NMR spectra are available for oligomersup to the hexamer, beyond which insufficient amounts of material areavailable. The trimer and the tetramer ¹H NMR spectra are in goodagreement with published spectra. See Hor, M. et al., Phytochemistry,1996, 42, for the trimer and tetramer and Sticher, O. J., Chromatogr. A,1999, 835, 59 for the trimer. In the case of the tetramer, partialthiolysis (see Hor et al.) establishes (4β,8)-regio- and-stereochemistry for the “upper” interflavan linkage, whereas the“lower” portion of the molecule is identical with the trimer. Thiscompound, in turn, has also been subjected to partial thiolysis, andboth interflavan linkages have been identified as (4β,8) (see Hor etal.). Since, therefore, in the chain extension process the first threeinterflavan linkages formed are exclusively of the 4β,8 type, the samemust be true for the additional interflavan linkages present in thelarger oligomers.

[0034] C. Cross Coupling of 3,5,7,3′,4′-Protected Monomers Having C-4(2-Benzothiazolyl)thio Groups and of A 3,5,7,3′,4′ Protected OligomerHaving a C-4 (2-Benzothiazolyl)thio and a 3,5,7,3′,4′-Protected Oligomer

[0035] 5,7,3′,4′-Tetra-O-benzylepicatechin monomers having2-(benzothiazolyl) thio groups at the C-4 position self-condensed in thepresence of silver tetrafluoraborate to yield a fairly complex mixturefrom which small amounts of the benzyl-protected,C₄-[(2-benzothiazolyl)thio]-dimer, -trimer, and presumably-tetramer canbe isolated. Also isolated is the rearranged benzyl-protected monomerand dimer where the group at the C-4 position is connected to thenitrogen rather than the sulfur of the thiazoyl ring. The migration ofthis moiety from sulfur to nitrogen is confirmed for the monomer by theobservation of a ¹³C NMR signal at δ 190.3 assignable to thethiocarbonyl carbon atom. The complexity of the above reaction mixtureis in part due to the formation of 4-O-3-linked oligomers similar to4-O-3-(5,7,3,4-tetra-O-benzyl)epicatechin. An attempt to avoid thereaction at the C-3 position by acetylating that position wasunsuccessful, resulting in low yields of the4-[(2-benzothiazolyl)thio]-substituted, protected oligomers (dimerthrough tetramer) and the formation hydroxylated oligomers.AgBF₄-induced self-condensation of3-O-acetyl-4-[(2-benzothiazolyl)thio]-5,7,3′,4′-tetra-O-benzylepicatechinresults in low yields of the 4-[(2-benzothiazolyl)thio]-substitutedoligomers, i.e., dimers, trimer and tetramer. Together with theseproducts, and in considerable quantities because of the small reactionscale, the 4-hydroxy by-products also formed. The by-products are3-O-acetyl,5,7,3′,4′-tetra-O-benzylepicatechin-(4β,8)-(3-O-acetyl-5,7,3′,4′-tetra-O-benzyl-4-hydroxyepicatechin,3-O-acetyl-5,7,3′,4′-tetra-O-benzylepicatechin-(4β,8)-(3-O-acetyl-5,7,3′,4′-tetra-O-benzylepicatechin)-(4β,8)-(3-O-acetyl-5,7,3′,4′-tetra-O-benzyl-4-hydroxyepicatechin,and 3-O-acetyl-5,7,3′,4′-tetra-O-benzylepicatechin-bis[(4β,8)-(3-O-acetyl-5,7,3′,4′-tetra-0-benzylepicatechin)]-(4β,8)-(3-O-acetyl-5,7,3′,4′-tetra-O-benzyl-4-hydroxyepicatechin)are formed.

[0036] Reaction ofbis(3-O-acetyl-5,7,3′,4′-tetra-O-benzyl)epicatechin-(4β,8)-[3-O-acetyl-4-[(2-benzothiozolyl)thio]-5,7,3′,4′-tetra-O-benzyepicatechinwith tetrakis (3-O-acetyl-5,7,3′,4′-tetra-O-benzyl) epicatechin(4β,8)₃-tetramer in the presence of silver tetrafluoroborate resulted inthe formation of the expected hexamer, i.e., hexakis(3-O-acetyl-5,7,3′,4′-tetra-O-benzyl)epicatechin (4β,8)₅-hexamer in 12%yield together with the by-products octakis(3-O-acetyl-5,7,3′,4′-tetra-O-benzyl) epicatechin (4β,8)₇-octamer,3-O-acetyl-5,7,3′,4′-tetra-O-benzylepicatechin-4β,8-(3-O-acetyl-5,7,3′,4′-tetra-O-benzyl-4-hydroxyepicatechin,and 3-O-acetyl-5,7,3′,4′-tetra-O-benzylepicatechin-bis[4β,8-(3-O-acetyl-5,7,3′,4′-tetra-O-benzylepicatechin)]-4β,8-(3-O-acetyl-5,7,3′,4′-benzylepicatechin)]-4β,8-(3-O-acetyl-5,7,3′,4′-tetra-O-benzyl-4-hydroxyepicatechin).

[0037] Thus, chain elongation can be performed in increments of twoflavanol units. This procedure should be of used for the chain-extensionof the larger protected epicatechin oligomers as these compounds farexceed the monomeric 5,7,3′,4′-protected, C-4 derivatized buildingblocks in value.

[0038] D. Chain Extension of Unprotected Monomers

[0039] The chain extension is carried out by reacting an unprotectedepicatechin or catechin monomer with 4-(benzylthio)epicatechin or4-(benzylthio)catechin monomer in the presence of dimethyl(methylthio)sulfonium tetrafluoroborate or preferably silver tetrafluoroborate. Theparticular virtue of this protocol resides in the exclusive formation of(4β,8)-interflavan linkages even for the unprotected substrates, whichwould otherwise yield mixtures of (4,6) and 4,8-linked products. On theother hand, the noxious nature of the benzyl mercaptan involved in thepreparation of the 4-(benzylthio) monomers is a considerable drawback.

[0040] Reagents, Test Procedures, and Analytical Procedures

[0041] Reagents

[0042] Pearlman's catalyst (20% Pd(OH)₂/C) was obtained from Aldrich andcontained up to 50% H₂O. Bentonite K-10 was purchased from Acros. Forother chemicals, see Tuickmantel, W. et al., J. Am. Chem. Soc., 1999,121, 12073.

[0043] Acetylation

[0044] Since the free procyanidins are poorly amenable to purificationbecause of their oxygen and acid sensitivity and their NMR spectra areuncharacteristic because of severe line broadening, these compounds arecharacterized as their peracetates. To avoid acid-induced interflavanbond cleavage during this reaction, the amount of pyridine in the aceticanhydride/pyridine reagent is two volumes of pyridine relative to theacetic anhydride, but as a result impurities emerge that eluteimmediately before the peracetates and which can not be removed on apreparative scale. The H¹ NMR spectra of the peracetates exhibit sharpsignals for two rotameters (in a 2:1 ratio for the trimer and in a 3:2ratio for all higher oligomers). The spectra up to the octamer are quitesuitable for compound identification. The acetate region serves as auseful “fingerprint”. ¹³C NMR spectra have been acquired for oligomersup to the hexamer.

[0045] The ¹H NMR spectra for the trimer and tetramer are in goodagreement with those published.

[0046] Spectra

[0047]¹H and ¹³C NMR spectra were acquired at nominal frequencies of 300and 75 MHz, respectively, in CDCl₃ unless specified otherwise. ¹H NMRspectra are referenced to internal TMS; ¹³C NMR spectra to internal TMSif so marked or otherwise to the CDCl₃ signal (δ 77.00). Combustionanalyses were carried out by Micro-Analysis, Inc. (Wilmington, Del.).

[0048] Column Chromatography

[0049] Column chromatography (CC) was carried out on Merck silica gel 60(No. 7734-7), particle size 63-200 μm. TLC: Merck silica gel 60 F₂₅₄(No. 7734-7), layer thickness 250 μm; visualization by UV light or withalkaline KMnO₄ solution.

[0050] High Pressure Liquid Chromatographic (HPLC) Analysis ofProcyanidins

[0051] Chromatographic analyses of free procyanidin oligomers wereperformed on a HP 1100 HPLC system (Hewlett Packard, Palo Alto, Calif.)equipped with an autoinjector, quaternary HPLC pump, column heater,diode array detector, fluorescence detector, and HP ChemStation for datacollection and sample manipulation. Normal phase separations wereperformed on a 250×4.6 mm Phenomenex (Torrance, Calif.) 5 μm Prodigycolumn. The detector was a fluorescence detector operating at λ_(ex)=276nm and λ_(em)=316 nm. The ternary mobile phase consisted of (A)dichloromethane, (B) methanol and (C) acetic acid:water (1:1 v/v).Separations were effected by a series of linear gradients of B into Awith a constant 4% C at a flow rate of 1 mL/minutes as follows: 0-30minutes, 14.0-28.4% B in A; 30-50 minutes, 28.4-38.0% B in A; 50-51minutes, 38.0-86.0% B in A; 51-56 minutes, 86.0% B in A isocratic.

[0052] HPLC: column A, Hewlett-Packard RP-8, 200×4.6 mm, at 1.0 mL/min;column B, Waters μBondapak C₁₈, 300×7.8 mm, at 2.8 mL/min; column C,Waters μBondapak C₁₈, 300×19 mm; column D, Waters μBondapak C₁₈, 300×30mm, at 42 mL/min; column E, Whatman Partisil 10, 500×9.4 mm, at 5.0mL/min; column F, Whatman Partisil 10, 500×22 mm, at 26 mL/min.Detection was by UV absorption at 280 nm. Retention times variedsubstantially depending on column history and other subtlecircumstances. They are quoted solely for orientation and should not beemployed for product identification without comparison to an authenticreference sample. See examples for further details.

[0053] High Pressure Liquid Chromatgraphic/Mass Spectra (HPLC/MS)Analysis of Procyanidins

[0054] HPLC/MS analyses of natural and synthetic procyanidins wereperformed on an HPLC system (as described above) which was interfaced toan HP Series 1100 mass selective detector (Model G1946A) equipped withan API-ES ionization chamber. Ionization reagents were added via a teein the eluent stream just prior to the mass spectrometer. Conditions foranalysis in the positive ion mode included the introduction of 0.05 Msodium chloride at a flow rate of 0.05 mL/minutes to assist ionization,a capillary voltage of 3.5 kV, a fragmentor voltage of 100 V, anebulizing pressure of 25 psig, and a drying gas temperature of 350° C.Scans were performed over a mass range of m/z 100-3000 at 1.96 s percycle.

[0055] Cell Lines

[0056] The human breast cancer cell lines MCF-7, SKBR-3, MDA 435, andMDA MB-231 were obtained from the Lombardi Cancer Center Cell CultureCore Facility at Georgetown University Medical Center. The MDA MB-231cell line was P53 defective, ER negative, and constitutively expressesK-ras. Cells were cultured in T-75s in IMEM medium (BioFluids Inc.)supplemented with 10% FBS (Gibco BRL Life Technologies) in a humidified5% CO₂ atmosphere at 37° C.

[0057] Cytotoxicity Assay

[0058] Cytotoxicity assays were performed on several human breast cancercell lines treated with test compounds in a 96 well microtiter plateformat using the microculture tetrazolium assay²⁸ modified for use withcrystal violet rather than MTT. Briefly, 1-2×10³ cells were added perwell and allowed to culture in a humidified, 5% CO₂ atmosphere untilthey reached approximately 50% confluence. Sterile filtered testcompounds were added at various concentrations, and the plates wereallowed to culture for an additional 12-36 hours. The growth medium wasthen removed, and each well was washed twice with 200 μL each of pH 7.4PBS. After washing, 50 μL of filtered crystal violet solution (2.5 g/125mL of methanol+375 mL of H₂O) was added. At the end of 5 minutes, thecrystal violet was removed, and the plate was washed three times withwater. Plates were allowed to dry, and the crystal violet stained cellswere resolubilized in 100 μL of 0.1 M sodium citrate in ethanol/water(1:1, v/v). At the end of 1 hour, the plates were scanned at 570 nm(ref. 405 nm) with a Molecular Devices Corporation microtiter platereader, and the data was recorded with the SOFTMAX software program. Theaverage of 3 readings was taken for each blank, control, vehicle, andtest concentration for statistical data manipulation.

[0059] Flow Cytometry

[0060] MDA MB-231 cells were cultured as described above until theyreach approximately 50% confluence. Sterile filtered test compounds orcatalase (Sigma # C9322) adjusted to 100 U/mL or heat inactivatedcatalase (solution immersed in boiling water for 15 min) or hydrogenperoxide (H₂O₂) was then added, and the cells were allowed to culturefor an additional 24 h. The cells were then trypsinized and counted, and1.5×10⁶ cells were taken for cell cycle analysis by the Vindelov method.See Vindelov, L. et al., “A Detergent Trypsin Method for the Preparationof Nuclei For Flow Cytometric DNA Analysis”, Cytometry, 1983, 3,323-327. Analyses were performed by the Lombardi Cancer Center FlowCytometry Core Facility at Georgetown University Medical Center.

[0061] Annexin V-FITC

[0062] The annexin V-FITC assay was performed on procyanidin-treated MDAMB-231 cells using the TACS™ Annexin V-FITC kit (Trevigen Inc.)according to the manufacturer's procedure.

EXAMPLES Example 1 Preparation of5,7,3′,4′-Tetra-O-benzyl-4-(2-hydroxyethoxy)epicatechin

[0063] To a solution of 21.5 g (33.0 mmol) of5,7,3′,4′-tetra-O-benzylepicatechin in 220 mL of anhydrous methylenechloride (CH₂Cl₂) was added at room temperature 11.0 mL (198 mmol) ofethylene glycol and then all at once with good stirring 15.0 g (66 mmol)of 2,3-dichloro-5,6-dicycano-1,4-benzoquinone (DDQ) was added. After 110minutes of vigorous stirring at room temperature under a calciumchloride (CaCl₂) tube, a solution of 8.5 g (69.5 mmol) of4-(dimethylamino)pyridine (DMAP) in 50 mL of anhydrous methylenechloride was added whereupon a copious dark precipitate appeared. Afteranother 10 minutes of stirring at room temperature, the mixture wasfiltered over a coarse glass frit, the precipitate was washed with 50 mLof methylene chloride, and the solution was evaporated to near dryness.The residue was filtered over silica gel (17×9 cm) with ethylacetate/hexane 1:1, and all product-containing fractions were pooled.After evaporation to approximately 75 mL, crystals began to appear(seeding may be necessary). An equal volume of hexane was added, andcrystallization was allowed to proceed at room temperature overnight.Suction filtration, washing twice with 25 mL of ethyl acetate/hexane(1:2), and drying in vacuo (initially at room temperature, then at 40°C.) furnished 10.6 g (45%) of5,7,3′,4′-tetra-O-benzyl-4-(2-hydroxyethoxy)epicatechin as an off-whitesolid. It was purified by HPLC to 97% (column B; 0-20 minutes, 50 to100% methylcyanide (CH₃CN) in H₂O, then CH₃CN; t_(R) 17.9 min).Additional product was obtained from the mother liquor by columnchromatography on silica (SiO₂) (33×5 cm) and elution with ethylacetate/hexane (1:2) (forerun), then 2:3 (product). After evaporation,the resulting amber glass (1.0 g, purity 69%) was crystallized twicefrom ethyl acetate/hexane to yield another 0.5 g (2%) of5,7,3′,4′-tetra-O-benzyl-4-(2-hydroxyethoxy)epicatechin (purity 98%).

Example 2 Condensation of 5 7,3′,4′-Tetra-O-benzylepicatechin with5,7,3′,4′-Tetra-O-benzyl-4-(2-hydroxyethoxy)epicatechin Catalyzed byAcidic Clay

[0064] To a solution/suspension of 9.26 g (14.2 mmol, 4 equiv.) of5,7,3′,4′-tetra-O-benzyl-epicatechin and 5.0 g of Bentonite K-10 clay in115 mL of anhydrous methylene chloride (CH₂Cl₂) was added, with icecooling, stirring and exclusion of moisture, within 2.5 hours 2.53 g(3.56 mmol) of 5,7,3′,4′-tetra-O-benzyl-4-(2-hydroxyethoxy)epicatechinin 35 mL of anhydrous methylene chloride. The bath temperature rose to+6° C. at the end of the addition. Stirring in the bath was continuedfor 1 hour, during which time the temperature rose to +12° C. The claywas filtered off with suction over celite, and the solids were washedtwo times with 50 mL of methylene chloride. Twenty mL of toluene wasadded, and the solution was evaporated to a small volume. The residuewas chromatographed on silica gel (60×5 cm) with ethylacetate/chloroform/hexane (1:14:14). Initially, 5.95 g of unreacted5,7,3′,4′-tetra-O-benzylepicathechin was eluted, followed by 4.01 g ofmonomer/dimer mixed fractions and 1.15 g of pure (98% by HPLC) bis(5,7,3′,4′-tetra-O-benzylepicatechin (4β,8)-dimer. The last traces ofthe dimer together with the trimer were eluted as a mixed fraction (0.27g) with a solvent ratio of 1:7:7.

[0065] The mixed fractions were each dissolved in methyl cyanide (CH₃CN)and separated by preparative HPLC (column D; 0-30 minutes, 80 to 100%(CH₃CN) in H₂O, then (CH₃CN); the retention times for the dimer andtrimer were 23.3 and 30.1 minutes, respectively. After combination ofthe appropriate fractions, evaporation, and drying in vacuo, thefollowing yields were obtained: 5,7,3′,4′ tetra-O-benzylepicatechin,6.89 g (74% recovery); bis(5,7,3′,4′-tetra-O-benzyl)epicatechin,(4β,8)-dimer, 4.26 g (92%); tris (5,7,3′,4′-tetra-O-benzyl)epicatechin(4β,8)₂-trimer 74 mg (2%): bis (5,7,3′,4′,-tetra-O-benzyl)-epicatechin(4β,8)-dimer. ¹³C NMR (CDCl₃, TMS) δ 158.34, 158.07, 157.91, 157.07,156.83, 156.56, 156.49, 155.89, 155.53, 155.07, 154.44, 152.83, 149.17,149.01, 148.92, 148.66, 148.60, 148.40, 148.18, 137.40, 137.38, 137.30,137.28, 137.22, 137.17, 137.01, 136.97, 132.61, 132.43, 131.18, 131.14,128.6-126.6, 119.96, 119.79, 118.79, 118.65, 115.02, 114.89, 114.35,114.05, 113.52, 112.93, 112.46, 111.58, 111.17, 104.45, 102.29, 101.76,94.34, 93.96, 93.33, 93.15, 92.93, 91.52, 78.84, 78.07, 75.63, 72.41,72.14, 71.48, 71.35, 71.22, 70.81, 70.48, 69.92, 69.86, 69.78, 69.47,69.05, 66.50, 65.15, 35.90, 35.78, 28.74, 28.61. Other data have beenpublished (see Part 1: Tuickmantel, W. et al. J. Am. Chem. Soc., 1999,121, 12073).

[0066] In another run (3.17 mmolar of5,7,3′,4′-tetra-O-benzyl-4-(2-hydroxyethoxy)-epicatechin), anessentially complete separation of monomer and dimer and of dimer andtrimer was achieved during column chromatography, with only the trimerand the very dilute tail of the dimer requiring purification by HPLC.The following yields were obtained: 5,7,3′,4′-tetra-O-benzylepicatechin,6.20 g (75% recovery); bis (5,7,3′,4′-tetra-O-benzyl)epicatechin(4β,8)-dimer, 3.63 g (88%); and tris(5,7,3′,4′-tetra-O-benzyl)epicatechin (4β,8)₂-trimer, 0.15 g (5%).

[0067] The HPLC analysis of the purified natural and synthetic oligomerswere compared. The purified natural procyanidin oligomers all exhibitedadditional peaks, with the number of additional peaks increasing as theoligomeric size increased.

Example 3 Condensation of Bis (5,7,3′,4′-tetra-O-benzyl)epicatechin(4β,8)-Dimer with5,7,3′,4′-Tetra-O-benzyl-4-(2-hydroxyethoxy)epicatechin Catalyzed byAcidic Clay

[0068] To a solution/suspension of 5.60 g (4.31 mmol, 3 equiv.) of bis(5,7,3′,4′-tetra-O-benzyl)epicatechin (4β,8)-dimer and 2.04 g ofBentonite K-10 clay in 45 mL of anhydrous methylene chloride (CH₂Cl₂)was added, with ice cooling, stirring and exclusion of moisture, within110 minutes 1.02 g (1.44 mmol) of5,7,3′,4′-tetra-O-benzyl-4-(2-hydroxyethoxy)-epicatechin in 15 mL ofanhydrous methylene chloride (CH₂Cl₂). The bath temperature rose to +6°C. at the end of the addition. Stirring in the bath was continued for 1hour, during which time the temperature rose to +12° C. The clay wasfiltered off with suction over celite, and the solids were washed fourtimes with 25 mL of ethyl acetate. The combined solutions wereevaporated. Attempted separation by column chromatography on silica gel(56×5 cm) with ethyl acetate/hexane/chloroform (1:10:10) failed toseparate the dimer and the trimer. Subsequent elution with a solventratio of 1:7:7 gave 0.50 g of a fraction consisting mostly of tetramertogether with residual trimer. The dimer/trimer fraction was againsubjected to column chromatography on silica gel (55×5 cm), this timestarting with ethyl acetate/chloroform/hexane 1:14:14. After elutionwith 20 L of this mixture, the solvent ratio was switched to 1:12:12 (5L), then 1:10:10, resulting in the recovery of 4.40 g of the dimer.Further elution with a mixing ratio of 1:8:8 gave 1.04 g of crude trimer(purity 90% by HPLC).

[0069] The crude trimer and the trimer/tetramer mixture were eachdissolved in methyl cyanide (CH₃CN) and separated by preparative HPLC(column D; 0-30 minutes, 80 to 100% CH₃CN in water, then CH₃CN); theretention times for the dimer, trimer, and tetramer were 22.5 (22.7),30.1 (30.8), and 33.9 minutes, respectively. After combination ofappropriate fractions, evaporation, and drying in vacuo, the followingyields were obtained: bis (5,7,3′,4′-tetra-O-benzyl)epicatechin(4β,8)-dimer, 4.43 g (79% recovery); tris(5,7,3′,4′-tetra-O-benzyl)epicatechin (4β,8)₂-trimer, 1.13 g (40%);tetrakis (5,7,3′,4′-tetra-O-benzyl)epicatechin (4β,8)₃-tetramer, 0.24 g(13%).

Example 4 Preparation of4-[(2-Benzothiazolyl)thio]-5,7,3′,4′-tetra-O-benzylepicatechin

[0070] To a solution of 6.5 g (39 mmol) of 2-mercaptobenzothiazole in 40mL of 1,2-dichloroethane [HPLC grade, filtered over basic alumina(activity I) immediately before use] was added dropwise in 10 minutesunder nitrogen with ice cooling and stirring 19.5 mL oftrimethylaluminum solution (2.0 M in toluene). The resulting ambersolution was stirred at 0° C. for 15 minutes, then a solution of 5.56 g(7.82 mmol) of 5,7,3′,4′,-tetra-O-benzyl-4-(2-hydroxyethoxy)epicatechinin 60 mL of 1,2-dichloroethane (pretreated as above) was added dropwisein 20 minutes. The orange-colored reaction mixture was stirred at roomtemperature for 5 hours, then cooled in an ice bath, and a solution of22.6 g (80 mmol) of potassium sodium tartrate tetrahydrate in 90 mL ofwater and 100 mL of 2.5 M aqueous sodium hydroxide was added dropwise(very cautiously at first because of gas evolution). Methylene chloride(100 mL) was added, and the phases were separated. The organic phase waswashed two times with 100 mL of 2.5 M aqueous sodium hydroxide and driedover sodium sulfate. After evaporation to a small volume, the residuewas chromatographed on a short silica column with ethyl acetate/toluene1:19 (until the beginning elution of product), then 1:9. The eluate wasevaporated to yield an oil, which soon turned into a light-yellow solid.This material was dissolved in 30 mL of hot ethyl acetate, 90 mL of1-chlorobutane was added, and the solution was seeded and set aside forcrystallization first at room temperature, then at −20° C. Theprecipitate was isolated by suction filtration, washed two times with 20mL of cold 1-chlorobutane, and dried in vacuo to yield 3.50 g of thepredominant diastereoisomer. Chromatography of the mother liquor(silica, ethyl acetate/methylene chloride/hexane 1:18:11 to 2:18:11)followed by crystallization from ethyl acetate/1-chlorobutane yielded anadditional 0.78 g of the major isomer (together 4.28 g, 67%) and 0.16 g(2.5%) of the less polar minor isomer.

[0071] Major diastereoisomer: mp 160-161° C. (fromethylacetate/1-chlorobutane); [α]_(D)+106°, [α]₅₄₆+133® (EtOAc, c 10.6gL⁻¹); ¹H NMR (CDCl₃) δ 7.89 (ddd, 1H, J=8, 1.2, 0.7 Hz), 7.78 (ddd, 1H,J=8, 1.2, 0.7 Hz), 7.47-7.20 (m, 19H), 7.17 (d, 1H, J=2 Hz), 7.12-7.00(m, 4H), 6.95 (B part of an ABq, 1H, J=8.5 Hz), 6.30, 6.29 (ABq, 2H, J=2Hz), 5.46 (d, 1H, J=2 Hz), 5.42 (s, 1H), 5.17 (s, 2H), 5.16 (s, 2H),5.10, 5.05 (ABq, 2H, J=12 Hz), 5.03 (s, 2H), 4.40 (ddd, 1H, J=6, 2.5, 1Hz), 2.00 (d, 1H, J=5.5 Hz); ¹³C NMR (CDCl₃) δ 165.00, 160.67, 158.76,155.95, 153.16, 148.96, 148.88, 137.17, 137.07, 136.53, 136.47, 135.29,130.76, 128.61, 128.45, 128.37, 128.16, 128.09, 127.75, 127.56, 127.49,127.46, 127.21, 126.57, 126.06, 124.41, 121.83, 120.97, 119.65, 114.91,113.58, 98.34, 94.48, 75.13, 71.32, 71.22, 70.78, 70.13, 69.86, 44.43;IR (film) 3554 (br), 1617, 1591, 1177, 1152, 1114, 735, 696 cm⁻¹.Analysis Calcd for C₅₀H₄₁NO₆S₂: C, 73.60; H, 5.06; N, 1.72. Found: C,73.92; H, 4.75; N, 1.74.

[0072] Minor diastereoisomer: mp 144-146° C. (from ethylacetate/1-chlorobutane); [α]_(D)−48.9°, [α]₅₄₆−64.6° (EtOAc, c 7.6gL⁻¹); ¹H NMR (CDCl₃) δ 7.79 (ddd, 1H, J=8, 1.2, 0.7 Hz), 7.66 (ddd, 1H,J=8, 1.2, 0.7 Hz), 7.47-7.25 (m, 14H), 7.17-7.11 (m, 2H), 7.08-6.89 (m,5H), 6.84-6.77 (m, 4H), 6.27, 6.25 (ABq, 2H, J=2 Hz), 5.45-5.40 (m, 2H),5.16 (narrow ABq, 2H), 5.11,5.07 (ABq, 2H, J=13 Hz), 5.07, 5.03 (ABq,2H, J=11.5 Hz), 4.94, 4.87 (ABq, 2H, J=11.5 Hz), 4.78 (q, 1H, J=5 Hz),4.39 (d, 1H, J=5 Hz); ¹H NMR (C₆D₆) δ 7.68 (d, 1H, J=8 Hz), 7.38 (d, 1H,J=2 Hz), 7.32-6.96 (m, 19H), 6.90-6.68 (m, 6H), 6.48 (d, 1H, J=2 Hz),6.22 (d, 1H, J=2.5 Hz), 5.82 (dd, 1H, J=5, 1.2 Hz), 5.57 (d, 1H, J=4.5Hz), 4.95 (s, 2H), 4.82 (q, 1H, J=4.5 Hz), 4.80 (s, 2H), 4.71 (s, 2H),4.70 (d, 1H, partly concealed), 4.58, 4.51 (ABq, 2H, J=12 Hz); ¹³C NMR(CDCl₃) δ 169.70, 160.84, 158.13, 155.45, 152.30, 148.53, 148.14,137.14, 137.02, 136.41, 135.88, 135.44, 129.67, 128.58, 128.36, 128.16,128.08, 127.87, 127.65, 127.55, 127.35, 127.31, 127.22, 127.00, 126.70,125.89, 124.15, 121.23, 120.85, 119.74, 114.41, 114.31, 100.87, 93.80,93.76, 76.46, 71.01, 70.73, 70.06, 70.00, 68.02, 46.51; IR (film) 3440(br), 1614, 1584, 1154, 1122, 752, 732, 696 cm⁻¹. Analysis: Calculatedfor C₅₀H₄₁NO₆S₂: C, 73.60; H, 5.06; N, 1.72. Found: C, 73.22; H, 4.64;N, 1.71.

[0073] The above reaction should be conducted in a well-ventilated fumehood because although 2-mercaptobenzothiazole is odorless, smallquantities of malodorous (but not very volatile) 2-(benzylthio)benzothiazole was formed in this reaction.

Example 5 Preparation of3-O-Acetyl-4-[(2-benzothiazolyl)thio]-5,7,3′,4′-tetra-O-benzylepicatechin

[0074] To a solution of 3.50 g (4.29 mmol) of 4-[(2-benzothiazolyl)thio]-5,7,3′,4′-tetra-O-benzylepicatechin (major diastereomer fromExample 4) and 53 mg (0.43 mmol) of 4-(dimethylamino)pyridine in 12 mLof anhydrous pyridine was added all at once 2.0 mL (21.5 mmol) of aceticanhydride. The reaction mixture was kept at room temperature in a closedflask for 50 hours. Ice and 150 mL of 5% aqueous hydrochloric acid wereadded. The product was extracted into 100+20 mL of methylene chloride.The combined organic phases were washed with 100 mL of water and twotimes with 50 mL of 10% aqueous sodium hydroxide; after each washing,the aqueous phase was back-extracted with 20 mL of methylene chloride.The combined organic phases were dried over magnesium sulfate andevaporated and the residue was taken up in a small volume of toluene andfiltered over silica with ethyl acetate/hexane (1:3). Evaporation anddrying in vacuo yielded 3.58 g (97%)3-O-acetyl-4-[(2-benzothiazolyl)thio]-5,7,3′,4′-tetra-O-benzylepicatechinas a yellowish foam: [α]_(D)+91.7°, [α]₅₄₆+115® (EtOAc, c 13.2 gL⁻¹); ¹HNMR (CDCl₃) δ 7.90 (d, 1H, J=8 Hz), 7.77 (d, 1H, J=8 Hz), 7.46-7.22 (m,19H), 7.11 (d, 1H, J=2 Hz), 7.09-7.00 (m, 3H), 6.99, 6.91 (ABq, 2H,J=8.5 Hz, A part d with J 2 Hz), 6.31, 6.30 (ABq, 2H, J=2.5 Hz), 5.63(dd, 1H, J=2.5, 1.2 Hz), 5.55 (s, 1H), 5.31 (d, 1H, J=2 Hz), 5.17,5.12(ABq, 2H, J=12 Hz), 5.14 (s, 2H), 5.10, 5.05 (ABq, 2H, J not readablebecause of overlap), 5.07, 5.02 (ABq, 2H, J=11.5 Hz), 1.84 (s, 3H); ¹³CNMR (CDCl₃, TMS) δ 169.08, 164.07, 160.69, 158.31, 156.03, 153.22,148.92, 148.89, 137.18, 137.16, 136.53, 136.31, 135.62, 130.29, 128.67,128.45, 128.24, 128.19, 127.78, 127.65, 127.43, 127.31, 126.87, 126.10,124.50, 122.16, 121.01, 119.80, 114.97, 113.51, 98.50, 94.46, 94.30,74.13, 71.44, 71.23, 70.74, 70.19, 70.13, 42.59, 20.84; IR 1750, 1616,1591, 1217, 1152, 1117, 734, 696 cm⁻¹. Analysis: Calculated forC₅₂H₄₃NO₇S₂: C, 72.79; H, 5.05; N, 1.63. Found: C, 73.01; H, 4.79; N,1.61.

Example 6 Preparation of Bis(3-O-acetyl-5,7,3′,4′-tetra-O-benzyl)epicatechin (4β,8)-Dimer

[0075] A solution of 1.5 mL (16 mmol) of acetic anhydride in 4 mL ofanhydrous pyridine was added all at once to 3.69 g (2.84 mmol) of bis(5,7,3′,4′-tetra-O-benzyl)epicatechin (4β,8)-dimer. The mixture wasoccasionally swirled until all starting material dissolved and thenallowed to stand in a closed flask at room temperature for 99 hours. Thereaction was terminated by addition of 30 mL of ethyl acetate and 2 mLof methanol and allowed to stand at room temperature for 1.5 hours.Another 20 mL of ethyl acetate was added. Then the solution was washedwith 200 mL of 0.5 M aqueous phosphoric acid (H₃PO₄). The aqueous layerwas back-extracted with 50 mL of ethyl acetate. The combined organicphases were dried over magnesium sulfate. After evaporation, the residuewas taken up in a small volume of toluene and chromatographed on a shortsilica column with ethyl acetate/hexane (1:9, then 1:3, finally 1:1).Evaporation and drying in vacuo yielded 3.82 g (97%) of bis(3-O-acetyl-5,7,3′,4′-tetra-O-benzyl)epicatechin (4β,8)-dimer as acolorless foam.

Example 7 Reaction of 3-O-Acetyl-4-[(2-benzothiazolyl)thio]-5,7,3′,4′-tetra-O-benzylepicatechin with Bis(3-O-acetyl-5,7,3′,4′-tetra-O-benzyl)epicatechin (4β,8)-Dimer

[0076] A 0.80 g (4.1 mmol) sample of silver tetrafluoroborate (AgBF₄)was dried in the reaction flask at 100° C. in an oil pump vacuum withexclusion of light for 1.5 hours. After cooling, the vacuum was brokenwith nitrogen, and a solution of 5.66 g (4.09 mmol) of bis(3-O-acetyl-5,7, 3′, 4′-tetra-O-benzyl)epicatechin (4β,8)-dimer in 60 mL of anhydroustetrahydrofuran was added all at once. The flask was placed in an icebath under dim light, and a solution of 1.40 g (1.64 mmol) of3-O-acetyl-4-[(2-benzothiazolyl)thio]-5,7,3′,4′-tetra-O-benzyleipcatechin in 30 mL of anhydroustetrahydrofuran was added dropwise in 70 minutes with stirring. Thereaction mixture turned yellow, and a turbidity eventually appeared.Stirring at 0° C. was continued for 40 minutes, during which time periodthe reaction mixture turned into a milky, whitish suspension.Triethylamine (1.1 mL, 8 mmol) was added, the mixture was evaporated tonear dryness, and the residue was filtered over a short silica columnwith ethyl acetate/hexane 1:1. The eluate was evaporated and the crudeproduct was analyzed by HPLC (column A; 0-30 minutes, 80 to 100% methylcyanide (CH₃CN) in water, then CH₃CN. The following peaks were observed(assignment/area %): t_(R) 5.0 (4-OH-monomer, 0.15), 12.6 (4-OH-dimer,0.25), 15.6 (dimer, 59.4), 24.8 (trimer, 23.4), 30.3 (tetramer, 12.5),33.3 (pentamer, 3.2), 35.4 (hexamer, 0.8), 37.3 (heptamer, 0.1), 39.1minutes (octamer, 0.02). A partial separation was achieved by columnchromatography on silica (38×9 cm). Initial elution with 25 L of ethylacetate/chloroform/hexane (1:10:9) did not result in product recovery(this stage was, however, essential for achieving separation). Another25 L of ethyl acetate/chloroform/hexane (1:11:8) eluted 4.01 g of thedimer (71% recovery; pure by HPLC). A fraction (1.72 g) consisting oftrimer, tetramer, and some pentamer was eluted with 20 L of ethylacetate/chloroform/hexane 1:12:7. Finally, the column was stripped withethyl acetate/chloroform/hexane (2:12:7) to give 0.87 g of a fractionconsisting mostly of the larger oligomers. The latter two fractions weretaken up in methyl cyanide (CH₃CN), and separated into several portionsby preparative HPLC (column D; 0-30 minutes, 80 to 100% (CH₃CN) inwater, then (CH₃CN), and the appropriate fractions were pooled and driedin vacuo to obtain the oligomers as colorless films or foams. Theretention times and yields relative to 3-O-acetyl-4-[(2-benzothiazolyl)thio]-5,7,3′,4′-tetra-O-benzylepicatechin for the trimer through octamerwere 31.9 minutes (1.46 g, 43%), 36.0 minutes (755 mg, 33%), 39.6minutes (204 mg, 11%), 45.0 minutes (45 mg, 2.6%), 52.8 minutes (13.8mg, 0.9%), and 64.1 minutes (5.2 mg, 0.3%), respectively; for the3-O-acetyl-5,7,3′,4′-tetra-O-benzylepicatechin-(4β,8)-(3-O-acetyl)-5,7,3′,4′-tetra-O-benzyl-4-hydroxyepicatechin,22.2 minutes (13.4 mg, 1.2%). The 4-OH monomer (i.e.,3-O-acetyl-5,7,3′,4′-tetra-O-benzyl 4-hydroxyepicatechin) was notrecovered from the silica column, probably because of its high polarity.The total mass balance relative to3-O-acetyl-4-[(2-benzothiazolyl)thio]-5,7,3′,4′-tetra-O-benzylepicatechinwas 92%.

Example 8 Coupling of3-O-Acetyl-4-[(2-benzothiazolyl)thio]-5,7,3′,4′-tetra-O-benzylepicatechinwith Tris (3-O-acetyl-5,7,3′,4′-tetra-O-benzyl)epicatechin(4β,8)₂-Trimer

[0077] The reaction was conducted analogously, to the coupling ofExample 7 using 0.41 g (2.1 mmol) of silver tetrafluoroborate (AgBF₄),4.40 g (2.12 mmol) of tris(3-O-acetyl-5,7,3′,4′-tetra-O-benzylepicatechin (4β,8)₂-trimer, and 729mg (850 μmol) of 3-O-acetyl-4-[2-(benzothiazolyl)thio]-5,7,3′,4′-tetra-O-benzylepicatechin. After filtration over silicawith ethyl acetate/hexane (1:1), the crude product was taken up inmethyl cyanide (CH₃CN) and separated into several portions bypreparative HPLC as above to yield the following products:3-O-acetyl-5,7,3′,4′-tetra-O-benzyl-4-hydroxyepicatechin (31 mg, 5%);3-O-acetyl-5,7,3′,4′-tetra-O-benzylepicatechin-(4β,8)-3-O-acetyl-5,7,3′,4′-tetra-O-benzyl-4-hydroxyepicatechin(34 mg, 6%); trimer (3.07 g, 70% recovery); tetramer (1.47 g, 62%);pentamer (221 mg, 15%); hexamer (57 mg, 5%); heptamer (25.2 mg, 2%);octamer (10.8 mg, 1%). Total mass balance relative to3-O-acetyl-4-[(2-benzothiazolyl)thio]-5,7,3′,4′-tetra-O-benzylepicatechin96%.

Example 9 Coupling of 3-O-Acetyl-4-[(2-benzothiazolyl)thio]-5,7,3′,4′-tetra-O-benzylepicatechin with Tetrakis(3-O-acetyl-5.7,3′,4′-tetra-O-benzyl) epicatechin (4(3,8)₃-Tetramer

[0078] The reaction was conducted analogously to the coupling ofExamples 7 and 8 using 0.34 g (1.75 mmol) of silver tetrafluoroborate(AgBF₄), 4.77 g (1.73 mmol) of tetrakis(3-O-acetyl-5,7,3′,4′-tetra-O-benzyl) epicatechin (4β,8)₃-tetramer, and592 mg (690 μmol) of 3-O-acetyl)-4-[(2-benzothiazolyl)thio]-5,7,3′,4′-tetra-O-benzylepicatechin. After filtration over silicawith ethyl acetate/hexane (1:1), the crude product was subjected inseveral portions to a preliminary separation by preparative HPLC (columnD; 0-30 minutes, 80 to 100% methyl cyanide (CH₃CN) in water; 30-38minutes, CH₃CN; 38-65 minutes, 10% ethyl acetyl in CH₃CN to yield thefollowing products: 3-O-acetyl-5,7,3′,4′-tetra-O-benzyl-4-hydroxyepicatechin (t_(R) 11.0 min; 19 mg, 4%);3-O-acetyl-5,7,31,4′-tetra-O-benzylepicatechin-(4β,8)₃-(3-O-acetyl-5,7,3′,4′-tetra-O-benzyl-4-hydroxyepicatechin(22.2 min; 47 mg, 10%); tetramer (36.0 min; 3.56 g, 74% recovery);pentamer (39.4 min; 1.03 g); hexamer (43.6 min; 260 mg); heptamer (46.0min; 86 mg); octamer (48.9 min; 41 mg); nonamer (52.2 min; 22 mg);decamer (56.2 min; 13.5 mg); undecamer (61.4 min; 8.2 mg). All productsfrom the pentamer on required additional purification because of peaktailing, which led to a contamination with lower oligomers thatincreased with the degree of oligomerization, and because of increasingcontamination with unidentified aliphatic material from the nonpolarsolvent and/or column. For sample preparation, a small percentage oftetrahydrofuran had to be added to the CH₃CN from the heptamer onbecause of limited solubility in CH₃CN alone. For the pentamer throughnonamer, the additional purification was performed on column D (0-30minutes, 80 to 100% CH₃CN in water, then CH₃CN. For the decamer andundecamer, column B was used in combination with the same gradient. Thenonamer, decamer, and undecamer still contained excessive amounts ofaliphatic impurities after this treatment and were subjected to a thirdHPLC purification on column A using the same gradient. The followingyields of pure products (97% or better by HPLC) were obtained: pentamer,987 mg (41%); hexamer, 226 mg (16%); heptamer, 68 mg (6.1%); octamer, 26mg (2.7%); nonamer, 11.5 mg (1.3%); decamer, 6.5 mg (0.8%); undecamer,2.5 mg (0.3%). Total mass balance relative to3-O-acetyl-4[(2-benzothiazoyl)thio]-5,7,3′,4′-tetra-O-benzylepicatechin82%.

Example 10 Hydrolysis of Acetyl-Protecting Groups from Acetyl-andBenzyl-Protected Oligomers

[0079] Part A—Tris (5,7,3′,4′-tetra-O-benzyl)epicatechin (4(3.8)₂-Trimer

[0080] To a solution of 1.54 g (742 μmol) of tris(3-O-acetyl-5,7,3′,4′-tetra-O-benzyl)epicatechin (4β,8)₂-trimer in 30 mLof tetrahydrofuran was added all at once 5.8 mL (8.9 mmol) of 40%aqueous tetra-n-butylammonium hydroxide. The reaction mixture wasallowed to stand at room temperature in a closed flask for 94 hours,then partially evaporated to remove the tetrahydrofuran. The residue wasdiluted with 20 mL of water, the product was extracted twice with 20 mLethyl acetate, and the combined organic phases were washed with 10 mL ofbrine and evaporated. Filtration over a short silica column with ethylacetate yielded, after evaporation and drying in vacuo, 1.44 g (99%) oftris (5,7,3′,4′-tetra-O-benzyl)epicatechin (4β,8)₂-trimer as a colorlessfoam:

[0081] Part B—Tetrakis (5,7,3′,4′-tetra-O-benzyl) epicatechin(4β,8)₃-Tetramer.

[0082] Reaction of 1.59 g (573 μmol) of tetrakis(3-O-acetyl-5,7,3′,4′-tetra-O-benzyl)epicatechin (4β,8)₃-tetramer with5.6 mL (8.6 mmol) of 40% aqueous tetra-n-butyl ammonium hydroxide in 29mL of tetrahydrofuran for 96 hours (as described for the trimer) yielded1.45 g (97%) of tetrakis (5,7,3′,4′-tetra-O-benzyl) epicatechin(4β,8)₃-tetramer as a colorless foam:

[0083] Part C—Pentakis (5,7,3′,4′-tetra-O-benzyl)epicatechin(4(0.8)₄-Pentamer.

[0084] Reaction of 1.81 g (524 μmol) of pentakis(3-O-acetyl-5,7,3′,4′-tetra-O-benzyl)-epicatechin (4β,8)₄-pentamer with6.9 mL (10.5 mmol) of 40% aqueous tetra-n-butyl ammonium hydroxide in 35mL of tetrahydrofuran for 118 hours, as described for the trimer,yielded 1.45 g (97%) of pentakis (5,7,3′,4′-tetra-O-benzyl)epicatechin(4β,8)₄-pentamer as a colorless foam. The analytical sample was furtherpurified by preparative HPLC (Column B, 0-30 min., 80-100% CH₃CN/H₂O,then CH₃,CN.

[0085] Part D—Hexakis (5,7,3°,4′-tetra-O-benzyl)epicatechin(4β,8)₅-Hexamer.

[0086] Reaction of 486 mg (117 μmol) ofhexakis(3-O-acetyl-5,7,3′,4′-tetra-O-benzyl)epicatechin (4β,8)₅-hexamerwith 1.5 mL (2.3 mmol) of 40% aqueous tetra-n-butyl ammonium hydroxidein 8 mL of tetrahydrofuran for 101 hours, (as described for the trimer),yielded 455 mg (100%) of hexakis (5,7,3′,4′-tetra-O-benzyl)epicatechin(4β,8)₅-hexamer as a colorless glass.

[0087] Part E—Heptakis (5,7,3′,4′-tetra-O-benzyl)epicatechin(4β,8)₆-Heptamer.

[0088] Reaction of 126 mg (26.1 μmol) of heptakis(3-O-acetyl-5,7,3′,4′-tetra-O-benzyl)epicatechin (4β,8)₆-heptamer with0.34 mL (0.52 mmol) of 40% aqueous tetra-n-butyl ammonium hydroxide in1.8 mL of tetrahydrofuran for 94 hours, (as described for the trimer),yielded 118 mg (100%) of heptakis (5,7,3′,4′-tetra-O-benzyl)epicatechin(4β,8)₆-heptamer as a colorless foam.

[0089] Part F—Octakis (5,7,3′,4′-tetra-O-benzyl)epicatechin(4β,8)₇-Octamer.

[0090] Reaction of 41.2 mg (26.1 μmol) ofoctakis(3-O-acetyl-5,7,3′,4′-tetra-O-benzyl)epicatechin (4β,8)₇ octamerwith 0.10 mL (0.15 mmol) of 40% aqueous tetra-n-butyl ammonium hydroxidein 0.5 mL of tetrahydrofuran for 126 hours (as described for the trimer)yielded 39.4 mg (102%) of octakis (5,7,3′,4′-tetra-O-benzyl)epicatechin(4β,8)₇-octamer as a colorless foam.

[0091] Part G—Nonakis (5,7,3′,4′-tetra-O-benzyl)epicatechin(4β,8)₈-Nonamer

[0092] Reaction of 17.9 mg (2.88 μmol) of nonakis(3-O-acetyl-5,7,3′,4′-tetra-O-benzyl)epicatechin (4β,8)₈-nonamer with 47μL (72 μmol) of 40% aqueous tetra-n-butyl ammonium hydroxide in 0.3 mLof tetrahydrofuran for 134 hours (as described for the trimer) yielded16.8 mg (100%) of nonakis (5,7,3′,4′-tetra-O-benzyl)epicatechin(4β,8)₈-nonamer as a colorless foam.

Example 11 Preparation of Epicatechin (4β,8)-Oligomers fromBenzyl-Protected Oligomers

[0093] A. Preparation of Epicatechin (4β,8)₂-Trimer

[0094] To a solution of 64.3 mg (33.0 μmol) ofbis(5,7,3′,4′-tetra-O-benzyl)epicatechin (4β,8)-dimer in 5 mL oftetrahydrofuran were added 5 mL of methanol, 0.25 mL of water, and 57 mgof 20% Pearlman's catalyst (Pd(OH)₂/C). The mixture was stirred under 1bar of hydrogen for 80 minutes and filtered over cotton. The filtrationresidue was washed two times with 10 mL of methanol. The combinedfiltrates were evaporated, and the residue was taken up in 10 mL of HPLCgrade water. The solution was filtered and lyophilized to yield 32.4 mg(101%) of epicatechin (4β,8)₂-trimer 6H₂O as a fluffy, amorphous,off-white solid: [α]_(D)+70.4°, [α]₅₄₆+84.40 (MeOH, c 2.2 gL⁻¹); (ref.4c: [α]_(D)+75.20, acetone, c 8.7 gL⁻¹; ref. 4d: [α]₅₇₈+90°, MeOH, c 2gL⁻¹; ref. 6: [α]_(D)+76.40, acetone, c 8.6 gL⁻¹; ref. 19b: [≢]₅₇₈+920,H₂O, c 1.9 gL⁻¹; ref. 19k: [α]_(D)+800, MeOH, c 1.6 gL⁻¹); ¹³C NMR(CD₃OD, TMS; δ 60-85 region only) δ 79.73, 77.08, 73.47, 72.94, 66.84;MS (API/ES) m/z 865.4 (100%; calcd for [M−H]⁻: 865.2), 577.0 (6%), 288.9(4%). Analysis: Calculated for C₄₅H₃₈O₁₈.6H₂O: C, 55.44; H, 5.17. Found:C, 55.71; H, 5.07.

[0095] B. Preparation of Epicatechin (4β,8)₃-Tetramer.

[0096] To a solution of 56 mg (21.6 μmol) oftris(5,7,3′,4′-tetra-O-benzyl)epicatechin (4β,8)₃-tetramer in 4 mL oftetrahydrofuran were added 4 mL of methanol, 0.2 mL of water, and 47 mgof 20% Pearlman's catalyst (Pd(OH)₂/C). The mixture was stirred under 1bar of hydrogen for 75 minutes and filtered over cotton. The filtrationresidue was washed two times with 5 mL of methanol. The combinedfiltrates were diluted with 5 mL of HPLC grade water and partiallyevaporated to remove the organic solvents. After dilution with another10 mL of HPLC grade water, the solution was filtered and lyophilized toyield 24.4 mg (89%) of epicatechin (4β,8)₃-tetramer.6H₂O as a fluffy,amorphous, off-white solid: [α]_(D)+93.30, [α]₅₄₆+114° (MeOH, c 9.3gL⁻¹) (ref. 4d: [α]₅₇₈+73.20, MeOH, c 3.7 gL⁻¹; ref. 4j: [α]_(D)+59.8°,acetone, c 12 gL⁻¹; ref. 6: [α]_(D)+109.50, acetone, c 12.3 gL⁻¹; ref.19i: [α]_(D)+89.2°, acetone, c 9 gL⁻¹; ref. 191: [α]_(D)+81°, MeOH, c1.1 gL⁻¹); MS (API/ES) m/z 1153.3 (55%; calcd for [M−H]⁻: 1153.3), 865.1(25%), 576.9 (100%), 500.1 (30%), 288.9 (4%). Analysis: Calculated forC₆₀H₅₀O_(240.6)H₂O: C, 56.96; H, 5.10. Found: C, 56.98; H, 4.83.

[0097] C. Epicatechin (4β,8)₄-Pentamer.

[0098] To a solution of 76 mg (23.4 μmol) of pentakis(5,7,3′,4′-tetra-O-benzyl)epicatechin (4β,8)₄-pentamer in 4 mLtetrahydrofuran were added 4 mL of methanol, 0.2 mL of water, and 60 mgof 20% Pearlman's catalyst (Pd(OH)₂/C). The mixture was stirred under 1bar of hydrogen for 2 hours and filtered over cotton. The filtrationresidue was washed with methanol, and the combined filtrates werepartially evaporated to remove the organic solvents. The residue wasdiluted with 10 mL of HPLC-grade water, filtered, and lyophilized toproduce 34.8 mg of epicatechin (4β,8)₄-pentamer as a fluffy, amorphous,off-white solid: [α]_(D)+116°, [α]₅₄₆+140° (methanol, c 8.3 gL⁻¹) (ref4d: [α]₅₇₈+96°, MeOH, c 1 gL⁻¹; ref. 19i: [≢]_(D)+102.10, acetone, c 10gL⁻¹; ref 191: [α]_(D)+102°, MeOH, c 1.2 gL⁻¹). Analysis: Calculated forC₇₅H₆₂O₃₀.7.5H₂O: C, 57.07; H, 4.92. Found: C, 56.99; H, 4.79.

[0099] D. Preparation of Epicatechin (4β,8)₅-Hexamer.

[0100] To a solution of 92.3 mg (23.7 μmol) of hexakis(5,7,3′,4′-tetra-O-benzyl)eplcatechin (4β,8)₅-hexamer in 8 mL oftetrahydrofuran were added 8 mL of methanol, 0.4 mL of water, and 169 mgof 20% Peariman's catalyst (Pd(OH)₂/C). The mixture was stirred under 1bar of hydrogen for 50 minutes and filtered over cotton. The filtrationresidue was washed with methanol, and the combined filtrates werepartially evaporated after addition of 10 mL of HPLC-grade water. Theresidue was diluted with another 20 mL of HPLC-grade water, filtered,and lyophilized to produce 47.4 mg of epicatechin (4β,8)-hexamer as afluffy, amorphous, off-white solid: [α]_(D)+123°, [α]₅₄₆+149° (methanol,c 8.6 gL⁻¹). Analysis: Calculated for C₉₀H₇₄O₃₆.9.2H₂O: C, 56.98; H,4.91. Found: C, 56.89; H, 4.61.

[0101] E. Preparation of Epicatechin (403.8)₆-Heptamer.

[0102] To a solution of 87.5 mg (19.3 μmol) of heptakis(5,7,3′,4′-tetra-O-benzyl)epicatechin (4β,8)₆-heptamer in 8 mL oftetrahydrofuron were added 8 mL of methanol, 0.4 mL of water, and 111 mgof 20% Pearlman's catalyst (Pd(OH)₂/C). The mixture was stirred under 1bar of hydrogen (H₂) for 1 hour and filtered over cotton. The filtrationresidue was washed with MeOH, and the combined filtrates were partiallyevaporated after addition of 10 mL of HPLC-grade H₂O. The residue wasdiluted with another 10 mL of HPLC-grade H₂O, filtered, and lyophilizedto produce 39.3 mg of epicatechin (4β,8)₆-heptamer as a fluffy,amorphous, off-white solid: [α]_(D)+134°, [α]₅₄₆+164° (MeOH, c 9.6gL⁻¹). Analysis: Calculated for C₁₀₅H₈₆O₄₂.10H₂O: C, 57.33; H, 4.86.Found: C, 57.49; H, 4.80

[0103] F. Preparation of Epicatechin (4β,8₇-Octamer.

[0104] To a solution of 35.7 mg (6.88 μmol) of octakis(5,7,3′,4′-tetra-O-benzyl)epicatechin (4β,8)₇-octamer in 3 mL oftetrahydrofuran were added 3 mL of methanol, 0.15 mL of water, and 57 mgof 20% Pearlman's catalyst (Pd(OH)₂/C). The mixture was stirred under 1bar of hydrogen for 55 minutes and filtered over cotton. The filtrationresidue was washed with methanol, and the combined filtrates werepartially evaporated after addition of 10 mL of HPLC-grade water. Theresidue was diluted with another 10 mL of HPLC-grade water, filtered,and lyophilized to produce 17.1 mg of epicatechin (4β,8)₇-octamer as afluffy, amorphous, off-white solid: [α]_(D)+148°, [α]₅₄₆+180° (methanol,c 5.2 gL⁻¹). Analysis: Calculated for C₁₂₀H₉₈O₄₈.10.7H₂O: C, 57.66; H,4.77. Found: C, 57.68; H, 4.79.

Example 12 Self-Condensation of4-[(2-Benzothiazolyl)thio]-5,7,3′,4′-tetra-O-benzylepicatechin Inducedby Silver Tetrafluoroborate

[0105] To a solution of 445 mg (545 μmol) of 4-[(2-benzothiazolyl)thio]-5,7,3′,4′-tetra-O-benzylepicatechin (the major diastereoisomer ofExample 4) in 5 mL of anhydrous tetrahydrofuran was added dropwisewithin 30 minutes in dim light and with magnetic stirring and icecooling a solution of 48 mg (247 μmol) of silver tetrafluoroborate(dried at 100° C. in an oil pump vacuum with exclusion of light for 110minutes immediately before use). Stirring at 0° C. was continued for 5minutes, then 0.2 mL of triethylamine was added. After evaporation, theresidue was prepurified by filtration over a short silica gel columnwith ethyl acetate/hexane (1:1) to yield 414 mg of a colorless foam. Thefive least polar major components of this complex mixture were isolatedby preparative HPLC (column D; 0-30 minutes, 80 to 100% methyl cyanide(CH₃CN) in water, then CH₃CN. The following retention times and yieldswere observed: 2-mercaptobenzothiazole, t_(R) 4.4 minutes, 19 mg;5,7,3′,4′-tetra-O-benzyl 4-(2-thioxobenzothiazol-3-yl)epicatechin, 15.4minutes, 18 mg (4%); starting monomer 21.4 minutes, 14 mg (3% recovery5,7,3′,4′-tetra-O-benzyl-epicatechin-(4β,8)-[5,7,3′,4′-tetra-O-benzyl-4-(2-thioxobenzothiazol-3-yl]epicatechin],23.5 minutes, 7 mg (2%);5,7,3′,4′-tetra-O-benzylepicatechin-(4β,8)-[4-(2-benzothiazolyl(thio)-5,7,3′,4′-tetra-O-benzylepicatechin],27.0 minutes, 15 mg (4%).

Example 13 Self-Condensation of 4-[(2-Benzothiazolyl)thio]-57,3′,4′-tetra-O-benzyl-epicatechin Induced by Acidic Clay

[0106] To a solution of 18.0 mg (21.0 μmol) of 4-[(2-benzothiazolyl)thio]-5,7,3′,4′-tetra-O-benzylepicatechin (major diastereoisomer inExample 4) in 1 mL of anhydrous methyl cyanide (CH₂Cl₂) was added 38 mga mortmorrolinite clay sold under the tradename Bentonite K-10. Themixture was stirred at room temperature for 160 minutes, filtered, andevaporated. The residue was separated by preparative HPLC (column B;0-30 minutes, 80 to 100% CH₃CN in water, then CH₃CN. The followingretention times and yields were observed: 2-mercaptobenzothiazole, t_(R)4.6 minutes, 0.6 mg; 5,7,3′,4′-tetra-O-benzyl4-(2-thioxobenzothiazol-3-yl)epicatechin, 13.2 minutes, 2.0 mg (11%);starting monomer, 19.2 minutes, 2.7 mg (15% recovery);5,7,3′,4′-tetra-O-benzylepicatechin-(4β,8)-[5,7,3′,4′-tetra-O-benzyl-4-(2-thioxobenzothiazol-3-yl)epicatechin,21.5 minutes, 0.6 mg (4%);5,7,3′,4′-tetra-O-benzylepicatechin-(4β,8)-[4-(2-benzothiazolyl)thio)-5,7,3′,4′-tetra-O-benzylepicatechin],25.9 minutes, 1.4 mg (9%);5,7,3′,4′-tetra-O-benzylepicatechin-(4β,8)-(5,7,3′,4′-tetra-O-benzylepicatechin)-(4β,8)-[4-((2-benzothiazolyl)thio)-5,7,3′,4′-tetra-O-benzylepicatechin],30.8 minutes, 1.2 mg (8%);5,7,3′,4′-tetra-O-benzylepicatechin-(4β,8)-(5,7,3′,4′-tetra-O-benzylepicatechin)-(4β,8)-[4-((2-benzothiazolyl)thio)-5,7,3′,4′-tetra-O-benzylepicatechin],34.2 minutes, 0.3 mg (2%).

Example 14 Self-Condensation of3-O-Acetyl-4-[(2-benzothiazolyl)thio]-5,7,3′,4′-tetra-O-benzylepicatechinInduced by Silver Tetrafluoroborate

[0107] To a solution of 355 mg (414 μmol) of3-O-acetyl-4-[(2-benzathiazolyl)thio]-5,7,3′,4′-tetra-O-benzylepicatechinin 4 mL of anhydrous tetrahydrofuran was added dropwise within 40minutes in dim light and with magnetic stirring and ice cooling asolution of 20 mg (103 μmol) of silver tetrafluoroborate dried at 90° C.in an oil pump vacuum with exclusion of light for 1 hour immediatelybefore use. Stirring at 0° C. was continued for 10 minutes, then 0.2 mLof triethylamine was added. After evaporation, the residue wasprepurified by filtration over a short silica gel column with ethylacetate/hexane (1:1) to yield 331 mg of a colorless foam. This mixturewas separated by preparative HPLC (column D; 0-30 minutes, 80 to 100%methyl cyanide (CH₃CN) in water, then CH₃CN. The following retentiontimes and yields were observed: 2-mercaptobenzothiazole, t_(R) 4.6 min.,6.4 mg; 3-O-acetyl-5,7,3′,4′-tetra-O-benzyl-4-hydroxyepicatechin, 11.1min., 13.2 mg (4.5%);3-O-acetyl-5,7,3′,4′-tetra-O-benzylepicatechin-(4β,8)-(3-O-acetyl-5,7,3′,4′-tetra-O-benzyl-4-hydroxyepicatechin),16.9 min., 38.3 mg (13%); starting material, 22.4 min., 156 mg. (44%); amixture of3-O-acetyl-5,7,3′,4′-tetra-O-benzylepicatechin-(4β,8)-[3-O-acetyl-4-[(2-benzothiazolyl)thio]-5,7,3′,4′-tetra-O-benzylepicatechin],and3-O-acetyl-5,7,3′,4′-tetra-O-benzylepicatechin-(4β,8)-(3-O-acetyl-5,7,3′,4′-tetra-O-benzylepicatechin)-(4β,8)-(3-O-acetyl-5,7,3′,4′-tetra-O-benzyl-4-hydroxyepicatechin):29.7 min., 54.3 mg; a mixture of3-O-acetyl-5,7,3′,4′-tetra-O-benzylepicatechin-(4β,8)-(3-O-acetyl-5,7,3′,4′-tetra-O-benzyl)epicatechin-(4β,8)-[3-O-acetyl-4-((2-benzothiazolyl)thio)-5,7,3′,4′-tetra-O-benzylepicatechin]and 3-O-acetyl-5,7,3′,4′-tetra-O-benzylepicatechin-bis[(4β,8)-(3-O-acetyl-5,7,3′,4′-tetra-O-benzylepicatechin)]-(4β,8)-(3-O-acetyl-5,7,3′,4′-tetra-O-benzyl-4-hydroxyepicatechin):34.6 min., 11.9 mg; 3-O-acetyl-5,7,3′,4′-tetra-O-benzylepicatechin-bis[(4β,8)-(3-O-acetyl-5,7,3′,4′-tetra-O-benzyl)epicatechin]-(4β,8)-[3-O-acetyl-4-((2-benzothiazolyl)thio)-5,7,3′,4′-tetra-O-benzylepicatechin]:38.9 min., 6.3 mg (2.1%). The mixture of3-O-acetyl-5,7,3′,4′-tetra-O-benzylepicatechin-(4β,8)-[3-O-acetyl-4[(2-benzothiazolyl)thio]-5,7,3′,4′-tetra-O-benzylepicatechin]and3-O-acetyl-5,7,3′,4′-tetra-O-benzylepicatechin-(4β,8)-(3-O-acetyl-5,7,3′,4′-tetra-O-benzylepicatechin)-(4β,8)-(3-O-acetyl-5,7,3′,4′-tetra-O-benzyl-4-hydroxyepicatechin)was separated by normal-phase HPLC (Column F; 0-40 min.,20 to 50% ethylacetate (EtOAC) in hexane, then 50%) its yield 43 mg (14%) of3-O-acetyl-5,7,3′,4′-tetra-O-benzylepicatechin-(4β,8)-[3-O-acetyl-4-[(2-benzothiazolyl)thio]-5,7,3′,4′-tetra-O-benzylepicatechin](t_(R) 22.1 min) and 6.4 mg. (2.2%) of3-O-acetyl-5,7,3′,4′-tetra-O-benzylepicatechin-(4β,8)-(3-O-acetyl-5,7,3′,4′-tetra-O-benzylepicateching)-4β,8-(30-acetyl-5,7,3′,4′-tetra-O-benzyl-4-hydroxyepicatechin)(t_(R) 32.8 min.). The mixture of3-O-acetyl-5,7,3′,4′-tetra-O-benzylepicatechin-(4β,8)-(3-O-acetyl-5,7,3′,4′-tetra-O-benzyl)epicatechin-(4β,8)-[3-O-acetyl-4-((2-benzothiazolyl)thio)-5,7,3′,4′-tetra-O-benzylepicatechin]and 3-O-acetyl-5,7,3′,4′-tetra-O-benzylepicatechin-bis[(4β,8)-(3-O-acetyl-5,7,3′,4′-tetra-O-benzylepicatechin)]-(4β,8)-(3-O-acetyl)-5,7,3′,4′-tetra-O-benzyl-4hydroxyepicatechin) was separated on column E using the same gradient toyield 5.4 mg (1.8%) of3-O-acetyl-5,7,3′,4′-tetra-O-benzylepicatechin-(4β,8)-(3-O-acetyl-5,7,3′,4′-tetra-O-benzyl)epicatechin-(4β,8)-[3-O-acetyl-4-((2-benzothiazolyl)thio)-5,7,3′,4′-tetra-O-benzylepicatechin](t_(R)34.8 min.) and 5.0 mg (1.7%) of3-O-acetyl-5,7,3′,4′-tetra-O-benzylepicatechin-bis[(4β,8)-(3-O-acetyl-5,7,3′,4′-tetra-O-benzylepicatechin)]-(4β,8)-(3-O-acetyl-5,7,3′,4′-tetra-O-benzyl-4-hydroxyepicatechin)(t_(R) 42.8 min.). For characterization, the products obtained by normalphase HPLC were repurified on column B (0-30 min., 80 to 100% methylcyanide (CH₃CN) in water then CH₃,CN).

Example 15 Reaction of3-O-Acetyl-5,7,3′-4′-tetra-O-benzylepicatechin-4,8-[3-O-acetyl-4-[(2-benzothiazolyl)thio]-5,7,3′,4′-tetra-O-benzylepicatechin]with Tetrakis (3-O-acetyl-5,7,3′,4′-tetra-O-benzyl)epicatechin(4β,8)₃-Tetramer

[0108] A 21 mg (0.11 mmol) sample of silver tetrafluoroborate was driedin the reaction flask at 100° C. in an oil pump vacuum with exclusion oflight for 1 hour. After cooling, the vacuum was broken with nitrogen,and a solution of 190 mg (68.8 μmol) of tetrakis(3-O-acetyl-5,7,3′,4′-tetra-O-benzyl)epicatechin (4β,8)₃-tetramer in 1mL of anhydrous tetrahydrofuran was added all at once. The flask wasplaced in an ice bath under dim light, and a solution of 35.5 mg (22.9μmol) of 3-O-acetyl-5,7,3′,4′-tetra-O-benzylepicatechin-(4β,8)[3-O-acetyl-4-[(2-benzothiazolyl)thio]-5,7,3′,4′-tetra-O-benzylepicatechin in 0.5 mL of anhydroustetrahydrofuran was added dropwise in 12 minutes with stirring. Stirringwas continued for 5 minutes at 0° C. and for 10 minutes at roomtemperature. Triethylamine (0.1 mL) was added, the mixture wasevaporated, and the residue was filtered over a short silica column withethyl acetate/hexane (1:1). The eluate was evaporated, and the crudeproduct mixture (230 mg) was separated by preparative HPLC (column D,280 mm; 0-30 min., 80 to 100% methyl cyanide (CH₃CN) in water, thenCH₃CN. The following retention times and yields were observed:3-O-acetyl-5,7,3′,4′-tetra-O-benzylepicatechin-(4β,8)-(3-O-acetyl-5,7,3′,4′-tetra-O-benzyl-4-hydroxyepicatechin),t_(R) 22.7 min., 21.0 mg (65%);3-O-acetyl-5,7,3′,4′-tetra-O-benzylepicatechin-bis[(4β,8)-(3-O-acetyl-5,7,3′,4′-tetra-O-benzylepicatechin)]-(4β,8)-(3-O-acetyl-5,7,3′,4′-tetra-O-benzyl-4-hydroxyepicatechin),34.6 min., 0.8 mg (2.5%);tetrakis(3-O-acetyl-5,7,3′,4′-tetra-O-benzyl)epicatechin(4β,8)₃-tetramer, 36.3 min., 176 mg (92.5% recovery);hexakis(3-O-acetyl-5,7,3′,4′-tetra-O-benzyl)epicatechin (4β,8)₅-hexamer,45.6 min., 11.7 mg (12%);octakis(3-O-acetyl-5,7,3′,4′-tetra-O-benzyl)epicatechin (4β,8)₇-octamer,1.4 mg (2.2%).

Example 16 Anticancer Activity

[0109] Cell cycle analysis of procyanidin-treated MDA MB-231 humanbreast cancer cells showed a G₀/G₁ arrest by the pentamer, no effect bythe dimer or trimer, and only a slight effect by the tetramer (See Table1). TABLE 1 Cell Cycle Analysis of MDA MB-231 Human Breast Cancer CellsTreated with Oligomeric Procyanidins Purified from Cocoa % G₀/G₁ % S %G₂/M Control 36.69 23.39 39.92 Vehicle 38.26 22.43 39.30 Dimer (200μg/mL; 24 hrs) 38.13 22.43 39.45 Control 42.28 35.61 22.12 Vehicle 43.6034.10 22.30 Trimer (200 μg/mL; 24 hrs) 43.22 35.98 20.80 Control 40.3336.25 23.42 Vehicle 43.71 34.42 21.87 Tetramer (200 μg/mL; 24 hrs) 51.4628.25 20.30 Control 38.33 21.05 40.61 Vehicle 37.84 21.39 40.77 Pentamer(200 μg/mL; 24 hrs) 66.03 17.23 16.67 Pentamer (200 μg/mL; 48 hrs) 88.316.07 5.62

[0110] The increase in G₀/G₁ was accompanied by a decrease of cellnumbers in the S phase and in G₂/M phase.

[0111] The manner of cell death (apoptosis or necrosis) was investigatedby the annexin V-fluorescein isothiocyanate (FITC) assay usingTrevigen's TACSTM Annexin V-FITC kit. Cell cycle analysis of MDA MB-231cells treated with natural and synthetic procyanidin trimer, tetramer,and pentamer is shown below. Flow cytometry of procyanidin-tested MDA MD231 human breast cover cells using annexin V-FITC and propidium iodide(control versus 24 hour treatment with 200 μg/mL of oligomer) is shownbelow. A is the epicatechin (4β,8)₂-trimer. B is the epicatechin(4β,8)₃-tetramer. C is the epicatechin (4β,8)₄-pentamer. The lower leftquadrant shows viable cells. The lower right quadrant shows earlyapoptic events. The upper right quadrant shows late apoptic events. Theupper left quadrant shows nonviable cells.

[0112] Cells treated with both natural and synthetic procyanidins showedsimilar profiles, with increases in cell populations in the upper rightquadrant being observed as the oligomer size increased. This quadrantrepresents annexin V positive cells that also take up propidium iodide,which cells are considered to be in either late apoptosis or innecrosis. The absence of a distinct cell population in the lower rightquadrant (cells associated with early apoptotic events) in the case ofpentamer-treated cells suggests a necrotic pathway to cell deathpossibly due to a direct interaction with the cell membrane leading todamage, cell crisis, and eventual death.

[0113] The pentamer-caused G₀/G₁ arrest was reversible in cells treatedup to 8 hours and irreversible after a 24 hour treatment. No differencein activity was observed between natural and synthetic procyanidintrimer. An approximately 15% increase in G₀/G₁ arrest was seen for asynthetic tetramer procyanidin compared to a natural procyanidintetramer. An approximately 30% increase for synthetic vs. naturalprocyanidin pentamer was shown. See Table 2 below. TABLE 2 ComparisonCell Cycle Analysis of MDA MB-231 Human Breast Cancer Cells Treated withNatural versus Synthetic Oligomeric Procyanidins % G₀/G₁ % S % G₂/MControl 28.65 49.28 22.06 Vehicle 27.19 49.61 23.2 Natural trimer (200μg/mL; 24 hrs) 28.46 48.49 23.05 Synthetic trimer (200 μg/mL; 24 hrs)26.98 49.57 23.45 Natural tetramer (200 μg/mL; 24 hrs) 36.82 43.37 19.02Synthetic tetramer (200 μg/mL; 24 hrs) 43.49 39.39 17.03 Naturalpentamer (200 μg/mL; 24 hrs) 45.99 38.25 15.76 Synthetic pentamer (200μg/mL; 24 hrs) 64.15 23.36 12.49

[0114] A recent report indicated that hydrogen peroxide (H₂O₂) wasartifactually produced in vitro by several different polyphenoliccompounds and was responsible for causing a variety of biologicalactivities. See Long, L. H. et al., Biochem. Biophys. Res. Commun.,2000, 273, 50. The results in Table 3 show that if hydrogen peroxide waspresent at the levels reported in the literature, it would produce ashift in the cell cycle to G₂/M with a decrease in G₀/G₁. The additionof catalase abrogated these effects, causing a shift in the cell cycleback to control values. The addition of catalase alone topentamer-treated cells produced no conclusive change in the cell cycleattributable to hydrogen peroxide, i.e., the typical G₀/G₁ arrest causedby the pentamer remained essentially unchanged.

[0115] To eliminate the possibility that the epicatechin(4β,8)₄-pentamer might inhibit catalase activity, hydrogen peroxide wasadded to pentamer-treated cells in the presence and absence of catalase.The addition of hydrogen peroxide to pentamer-treated cells led to anincrease in G₀/G₁ and G₂/M arrest at the expense of cells in the Sphase. Catalase addition caused a shift back to the G₀/G₁ arrest typicalof pentamer-treated cells, and heat-inactivated catalase had no effect.Thus, the G₀/G₁ arrest was directly caused by the pentamer, not by thehydrogen peroxide. These differences can be attributed to the higherpurities of the synthetic procyanidins. TABLE 3 Cell Cycle Analysis ofMDA MB-231 Pentamer Treated Cells % G₀/G₁ % S % G₂/M Control 33.14 44.0622.81 Vehicle 36.44 41.63 21.94 100 μM H₂O₂; 24 hrs 20.32 44.92 34.76100 μM H₂O₂ + catalase; 24 hrs 35.20 42.93 21.86 100 μM H₂O₂ + heatinactivated 20.27 45.48 34.25 catalase; 24 hrs Control 29.87 46.21 23.92Vehicle 30.28 47.25 22.47 Pentamers (200 μg/mL); 24 hrs 44.94 38.0117.05 Pentamers (200 μg/mL) + catalase; 24 hrs 41.23 39.65 20.12Pentamers (200 μg/mL) + heat inactivated 42.89 39.43 17.68 catalase; 24hrs Pentamers (200 μg/mL) + 100 μM H₂O₂; 42.67 18.63 38.71 24 hrsPentamers (200 μg/mL) + 100 μM H₂O₂ + 48.20 31.12 20.68 catalase; 24 hrsPentamers (200 μg/mL) + 100 μM H₂O₂ + 39.47 23.39 37.14 heat inactivatedcatalase; 24 hrs

[0116] Collectively, the above results confirm the cytotoxicity to humanbreast cancer cell lines by an epicatechin pentamer, whether purifiedfrom cocoa polyphenol extracts or prepared synthetically. Theprocyanidin pentamer caused a G₀/G₁ arrest in MDA MB-231 cells which wasindependent of any effects caused by hydrogen peroxide. An increase inannexin V and propidium iodide positive cells suggests that thepentamer-treated cells quickly entered into a necrotic phase of celldeath.

[0117] The above examples are merely illustrative and no limitation ofthe preferred embodiments is implied. The skilled artisan will recognizemany variations without departing from the spirit and scope of theinvention.

What is claimed is:
 1. A process for preparing a mixture of a 5,7,3′,4′-tetra-O-protected epicatechin (4β,8)-dimer and other oligomers comprises the step of coupling a 5,7,3′,4′-tetra-O-protected epicatechin monomer with a 5,7,3′,4′-tetra-O-protected-4-acyloxy epicatechin monomer in the presence of an acidic clay.
 2. A process for preparing a mixture of 5,7,3′,4′-tetra-O-protected epicatechin (4β,8)-oligomers comprises the step of coupling a 5,7,3′,4′-tetra-O-protected epicatechin (4β,8)-oligomer with a 5,7,3′,4′-tetra-O-protected-4-acyloxy epicatechin monomer in the presence of an acidic clay.
 3. The process of claim 1 or 2, wherein the acidic clay is a mortmorillonite clay.
 4. The process of claim 1, wherein the protecting groups on the protected monomers are protecting groups which do not deactivate the A ring of the protected monomers.
 5. The process of claim 2, wherein the protecting groups on the protected oligomer are protecting groups that do not deactivate the A ring of the upper mer of the oligomer and the protecting groups on the protected monomer are protecting groups that do not deactivate that A ring of the monomer.
 6. The process of claim 4 or 5, wherein the protecting groups are benzyl groups.
 7. The process of claim 1 or 2, wherein the 4-acyloxy group is a C₂-C₆ alkoxy group having a terminal hydroxyl group.
 8. The process of claim 7, wherein the C₂-C₆ alkoxy group having the terminal hydroxyl group is a 2-hydroxyethoxy group.
 9. The process of claim 1, wherein the protected monomers are 5,7,3′,4′-tetra-O-benzylepicatechin and 5,7,3′,4′-tetra—benzyl-4-[2-hydroxyethoxy] epicatechin; wherein the mixture comprises the benzyl-protected epicatechin (4β,8)-dimer and benzyl-protected epicatechin (4β,8)₂-trimer.
 10. The process of claim 9, wherein the benzyl-protected epicatechin (4β,8)-dimer is the major product in the mixture.
 11. The process of claim 2, wherein the oligomer is a benzyl-protected (4β,8)-dimer; wherein the monomer is 5,7,3′,4′-tetra-O-benzyl-4-[2-hydroxyethoxy]epicatechin; and wherein the mixture comprises a benzyl-protected epicatechin (4β,8)-dimer, a (4β,8)₂-trimer, and a (4β,8)₃-tetramer.
 12. The process of claim 1, further comprising the step of separating the protected dimer and other protected oligomers from the monomer by column chromatography.
 13. The process of claim 2, further comprising the step of separating the protected oligomers and protected monomer by column chromatography.
 14. The process of claim 12 or 13, further comprising the step of replacing the protecting groups on the separated dimer or oligomers with hydrogen.
 15. A process for preparing a mixture of benzyl-protected (4β, 8)-oligomers of epicatechin or catechin comprises reacting a 5,7,3′,4′-tetra-O-benzyl-protected epicatechin or catechin monomer or a 5,7,3′,4′-tetra-O-benzyl-protected (4β,8)-epicatechin or catechin oligomer and 3-O-acetyl-4-[(2-benzothiazolyl)thio]-5,7,3′,4′-tetra-O-benzylepicatechin in the presence of silver tetrafluoroborate.
 16. A process for preparing a mixture of acetyl-protected and benzyl-protected (4β,8)-oligomers of epicatchin or catechin comprises reacting a 3-O-acetyl-5,7,3′,4′-tetra-O-benzylepicatechin monomer or a 3-O-acetyl-5,7,3′,4′-tetra-O-benzylepicatechin (4β,8)-oligomer and 3-O-acetyl-4-[(2-benzothiazolyl)thio]-5,7,3′,4′-tetra-O-benzylepicatechin in the presence of silver tetrafluoroborate.
 17. The process of claim 15 or 16, wherein the silver tetrafluoroborate is dried before the reaction.
 18. The process of claim 17, wherein the drying is vacuum drying carried out immediately before the reaction.
 19. The process of claim 16, wherein the mixture comprises protected trimers through protected octamers.
 20. The process of claim 15 or 16, further comprising the step of isolating the protected oligomers in the mixture by reverse phase high pressure liquid chromatography.
 21. The process of claim 20, further comprising the step of removing the acetyl-protecting group(s) from the isolated oligomers.
 22. The process of claim 21, wherein the acetyl group(s) removal is carried out with aqueous tetra-n-butyl ammonium hydroxide.
 23. The process of claim 20, further comprising the step of removing the benzyl-protecting groups from the isolated oligomers.
 24. The process of claim 23, wherein the benzyl groups removal is carried out by hydrogenolysis.
 25. The process of claim 20, further comprising the steps of removing the acetyl protecting group(s) and then removing the benzyl protecting groups from the isolated oligomers.
 26. The process of claim 25, wherein the acetyl group(s) removal is carried out with aqueous tetra-n-butyl ammonium hydroxide and the benzyl groups removal is carried out by hydrogenolysis.
 27. A process for preparing a mixture of 5,7,3′,4′-tetra-O-benzyl (4β,8)-oligomers comprises the steps of: (a) activating with 2-(benzothiazolyl)thio groups the C-4 positions of each of epicatechin 5,7,3′,4′-tetra-O-benzylepicatechin; and (b) self condensing the activated, protected monomers in the presence of silver tetrafluoroborate or an acidic clay to form a benzyl-protected condensed epicatechin (4β,8)-oligomer.
 28. The process of claim 27, further comprising the steps of separating the protected dimer, trimer, and tetramer removing the benzyl protecting groups.
 29. A process for chain extending protected epicatechin (4β,8)-oligomers comprises the step of condensing an epicatechin (4β,8) oligomer having 3-O-acetyl protecting groups and 5,7,3′,4′-tetra-O-benzyl protecting groups on all mers and a C-4-[2(benzothiazolyl)thio] activating group on a terminal mer with an epicatechin oligomer having 3-O-acetyl and 5,7,3′,4′-tetra-O-benzyl protecting groups on each mer in the presence of silver tetrafluoroborate or an acidic clay.
 30. The process of claim 29, wherein one of the C-4 activated, protected oligomers is a 3-O-acetyl-5,7,3′,4′-tetra-O-benzylepicatechin-(4β,8)-[3-O-acetyl-4-[(2-benzothiozolyl)thio-5,7,3′,4′-tetra-O-benzylepicatechin]; wherein the benzyl-protected oligomer is tetrakis (3-O-acetyl-5,7,3′,4′-tetra-O-benzyl)epicatechin (4β,8), wherein the protected, chain-extended oligomer is hexakis (3-O-acetyl-5,7,3′,4′-tetra-O-benzyl tetramer epicatechin) (4β,8)₅-hexamer.
 31. 4-[(2-Benothiazolyl)thio]-5,7,3′,4′-tetra-O-benzylepicatechin or 4-[(2-benzothiazolyl)thio]-5,7,3′,4′-tetra-O-benzylcatechin.
 32. A process for preparing the compound of claim 31 comprises reacting 5,7,3′,4′-tetra-O-benzyl-4-(2-hydroxyethoxy)epicatechin or 5,7,3′,4′-tetra-O-benzyl-4-(2-hydroxyethoxy)catechin with an organoaluminum thiolate generated from 2-mercaptobenzothiazole.
 33. 4-[(2-Benzothiazolyl)thio]-3-O-acetyl-5,7,3′,4′-tetra-O-benzylepicatechin or 4-[(2-benzothiazolyl)thio]-3-O-acetyl-5,7,3′,4′-tetra-O-benzylcatechin.
 34. A process for preparing the compound of claim 33 comprises reacting 5,7,3′,4′-tetra-O-benzyl-4-(2-hydroxyethoxy)epicatechin or 5,7,3′,4′-tetra-O-benzyl-4-(2-hydroxyethoxy)catechin with an organoaluminum thiolate generated from 2-mercaptobenzothiazole followed by acetylation.
 35. A method of treating breast cancer in a mammal in need of such treatment, which treatment inhibits cancer cell growth through cell cycle arrest in the G₀/G phase and comprises administering to the mammal epicatechin-(4β,8)₄-pentamer, wherein the breast cancer cells are selected from the group consisting of human breast cancer cell lines MCF-7, SKBR-3, MDA 435, and MDA MB-231.
 36. The method of claim 35 wherein the pentamer is a purified procyanidin fraction isolated from cocoa beans as a cocoa extract.
 37. The method of claim 36, wherein the pentamer is a synthetically prepared procyanidin. 